Compositions and Methods for Inhibiting Expression of Eg5 and VEGF Genes

ABSTRACT

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a SNALP formulation, and methods of using the compositions to inhibit the expression of the Eg5 and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2009/036233, filed Mar. 5, 2009 which claims the benefit of U.S.Provisional Application No. 61/034,019, filed Mar. 5, 2008, and U.S.Provisional Application No. 61/083,367, filed Jul. 24, 2008, and U.S.Provisional Application No. 61/086,381, filed Aug. 5, 2008, and U.S.Provisional Application No. 61/112,079, filed Nov. 6, 2008, and U.S.Provisional Application No. 61/150,664, filed Feb. 6, 2009 which arehereby incorporated in their entirety by reference.

FIELD OF THE INVENTION

This invention relates to compositions containing double-strandedribonucleic acid (dsRNA), and their use in mediating RNA interference toinhibit the expression of a combination of genes, e.g., the Eg5 andVascular Endothelial Growth Factor (VEGF) genes formulated in SNALP, andthe use of the compositions to treat pathological processes mediated byEg5 and VEGF expression, such as cancer.

BACKGROUND OF THE INVENTION

The maintenance of cell populations within an organism is governed bythe cellular processes of cell division and programmed cell death.Within normal cells, the cellular events associated with the initiationand completion of each process is highly regulated. In proliferativedisease such as cancer, one or both of these processes may be perturbed.For example, a cancer cell may have lost its regulation (checkpointcontrol) of the cell division cycle through either the overexpression ofa positive regulator or the loss of a negative regulator, perhaps bymutation.

Alternatively, a cancer cell may have lost the ability to undergoprogrammed cell death through the overexpression of a negativeregulator. Hence, there is a need to develop new chemotherapeutic drugsthat will restore the processes of checkpoint control and programmedcell death to cancerous cells.

One approach to the treatment of human cancers is to target a proteinthat is essential for cell cycle progression. In order for the cellcycle to proceed from one phase to the next, certain prerequisite eventsmust be completed. There are checkpoints within the cell cycle thatenforce the proper order of events and phases. One such checkpoint isthe spindle checkpoint that occurs during the metaphase stage ofmitosis. Small molecules that target proteins with essential functionsin mitosis may initiate the spindle checkpoint to arrest cells inmitosis. Of the small molecules that arrest cells in mitosis, thosewhich display anti-tumor activity in the clinic also induce apoptosis,the morphological changes associated with programmed cell death. Aneffective chemotherapeutic for the treatment of cancer may thus be onewhich induces checkpoint control and programmed cell death.Unfortunately, there are few compounds available for controlling theseprocesses within the cell. Most compounds known to cause mitotic arrestand apoptosis act as tubulin binding agents. These compounds alter thedynamic instability of microtubules and indirectly alter thefunction/structure of the mitotic spindle thereby causing mitoticarrest. Because most of these compounds specifically target the tubulinprotein which is a component of all microtubules, they may also affectone or more of the numerous normal cellular processes in whichmicrotubules have a role. Hence, there is also a need for agents thatmore specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that are localized tothe mitotic spindle and known to be required for formation and/orfunction of the bipolar mitotic spindle. Recently, there was a report ofa small molecule that disturbs bipolarity of the mitotic spindle (Mayer,T. U. et. al. 1999. Science 286 (5441) 971-4, herein incorporated byreference). More specifically, the small molecule induced the formationof an aberrant mitotic spindle wherein a monoastral array ofmicrotubules emanated from a central pair of centrosomes, withchromosomes attached to the distal ends of the microtubules. The smallmolecule was dubbed “monastrol” after the monoastral array. Thismonoastral array phenotype had been previously observed in mitotic cellsthat were immunodepleted of the Eg5 motor protein. This distinctivemonoastral array phenotype facilitated identification of monastrol as apotential inhibitor of Eg5. Indeed, monastrol was further shown toinhibit the Eg5 motor-driven motility of microtubules in an in vitroassay. The Eg5 inhibitor monastrol had no apparent effect upon therelated kinesin motor or upon the motor(s) responsible for golgiapparatus movement within the cell. Cells that display the monoastralarray phenotype either through immunodepletion of Eg5 or monastrolinhibition of Eg5 arrest in M-phase of the cell cycle. However, themitotic arrest induced by either immunodepletion or inhibition of Eg5 istransient (Kapoor, T. M., 2000. J Cell Biol 150 (5) 975-80). Both themonoastral array phenotype and the cell cycle arrest in mitosis inducedby monastrol are reversible. Cells recover to form a normal bipolarmitotic spindle, to complete mitosis and to proceed through the cellcycle and normal cell proliferation. These data suggest that aninhibitor of Eg5 which induced a transient mitotic arrest may not beeffective for the treatment of cancer cell proliferation. Nonetheless,the discovery that monastrol causes mitotic arrest is intriguing andhence there is a need to further study and identify compounds which canbe used to modulate the Eg5 motor protein in a manner that would beeffective in the treatment of human cancers. There is also a need toexplore the use of these compounds in combination with otherantineoplastic agents.

VEGF (also known as vascular permeability factor, VPF) is amultifunctional cytokine that stimulates angiogenesis, epithelial cellproliferation, and endothelial cell survival. VEGF can be produced by awide variety of tissues, and its overexpression or aberrant expressioncan result in a variety disorders, including cancers and retinaldisorders such as age-related macular degeneration and other angiogenicdisorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

SUMMARY OF THE INVENTION

Disclosed are compositions having two double-stranded ribonucleic acids(dsRNA) for inhibiting the expression of a human kinesin family member11 (Eg5/KSP) and a human VEGF gene in a cell. The dsRNAs are formulatedin a stable nucleic acid lipid particle (SNALP). Also disclosed aremethod for using the composition to decrease expression of Eg5/KSPand/or VEGF in a cell, and method of treatment of a disease, e.g., livercancer, using the compositions of the invention.

Accordingly, disclosed herein is a composition having a firstdouble-stranded ribonucleic acid (dsRNA) for inhibiting the expressionof a human kinesin family member 11 (Eg5/KSP) gene in a cell and asecond dsRNA for inhibiting expression of a human VEGF in a cell,wherein both said first and said second dsRNA are formulated in a stablenucleic acid lipid particle (SNALP); said first dsRNA consists of afirst sense strand and a first antisense strand, and said first sensestrand has a first sequence and said first antisense strand has a secondsequence complementary to at least 15 contiguous nucleotides of SEQ IDNO:1311 (5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence iscomplementary to said second sequence and wherein said first dsRNA isbetween 15 and 30 base pairs in length; and said second dsRNA consistsof a second sense strand and a second antisense strand, said secondsense strand having a third sequence and said second antisense strandhaving a fourth sequence complementary to at least 15 contiguousnucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), whereinsaid third sequence is complementary to said fourth sequence and whereineach strand is between 15 and 30 base pairs in length.

In some embodiments, the first antisense strand has a second sequencecomplementary to SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′) and thesecond antisense strand has a fourth sequence complementary to SEQ IDNO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′). In other embodiments, the firstdsRNA consists of a sense strand consisting of SEQ ID NO:1534(5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQID NO:1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consistsof a sense strand consisting of SEQ ID NO:1536(5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQID NO:1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′). In further embodiments, eachstrand is modified as follows to include a 2′-O-methyl ribonucleotide asindicated by a lower case letter “c” or “u” and a phosphorothioate asindicated by a lower case letter “s”: the first dsRNA consists of asense strand consisting of SEQ ID NO:1240(5′-ucGAGAAucuAAAcuAAcuTsT-3′), and an antisense strand consisting ofSEQ ID NO:1241 (5′-AGUuAGUUuAGAUUCUCGATsT); the second dsRNA consists ofa sense strand consisting of SEQ ID NO:1242(5′-GcAcAuAGGAGAGAuGAGCUsU-3′) and an antisense strand consisting of SEQID NO:1243 (5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).

In some embodiments, the first dsRNA contains two overhangs and thesecond dsRNA contains an overhang at the 3′ of the antisense and a bluntend at the 5′ end of the antisense strand.

The first and second dsRNA can have at least one modified nucleotide.For example, each dsRNA can have at least one modified nucleotide chosenfrom the group of: a 2′-O-methyl modified nucleotide, a nucleotidehaving a 5′-phosphorothioate group, and a terminal nucleotide linked toa cholesteryl derivative or dodecanoic acid bisdecylamide group. Themodified nucleotide can be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base having nucleotide. In some embodiments, the firstand second dsRNA each comprise at least one 2′-O-methyl modifiedribonucleotide and at least one nucleotide having a 5′-phosphorothioategroup.

Each strand of each dsRNA can be, e.g., 19-23 bases in length, or,alternatively 21-23 bases in length. In one embodiment, each strand ofthe first dsRNA is 21 bases in length and the sense strand of the seconddsRNA is 21 bases in length and the antisense strand of the second dsRNAis 23 bases in length.

In some embodiments, the first and second dsRNA are present in anequimolar ratio.

As described herein, the dsRNAs are formulated as SNALPS. In someembodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC,and PEG2000-C-DMA. For example, the SNALP can have the components in theproportions listed in Table 17.

The composition of the invention can be used to reduce expression of Eg5and/or VAGF. In some embodiments, the composition of the invention, uponcontact with a cell expressing Eg5, inhibits expression of Eg5 by atleast 40, 50, 60, 70, 80, or by at least 90%. In other embodiments, thecomposition of the invention, upon contact with a cell expressing VEGF,inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by atleast 90%. Administration of the composition to a cell can expression ofboth Eg5 and VEGF in said cell. The composition of claims 1-17, whereinthe composition is administered in a nM concentration.

Administration of the composition of the invention to a cell can resultin, e.g., an increase in mono-aster formation in the cell.Administration of the composition to a mammal can result in at least oneeffect selected from the group consisting of prevention of tumor growth,reduction in tumor growth, or prolonged survival in said mammal. Theeffect can be measured using at least one assay selected from the groupconsisting of determination of body weight, determination of organweight, visual inspection, mRNA analysis, serum AFP analysis andsurvival monitoring. Included are compositions with these effect whenadministered in a nM concentration.

In a further embodiment the composition of the invention includesSorafenib.

Also included in the invention are methods of suing the compositions ofthe invention. In one embodiment is are methods for inhibiting theexpression of Eg5/KSP and VEGF in a cell by administering any of thecompositions of the invention to the cell. Other embodiments are methodsfor preventing tumor growth, reducing tumor growth, or prolongingsurvival in a mammal in need of treatment for cancer by administeringthe composition to said mammal. In some embodiments the mammal has livercancer, e.g., the mammal is a human with liver cancer. The method caninclude a further step of administering Sorafenib.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing liver weights as percentage of body weightfollowing administration of SNALP-siRNAs in a Hep3B mouse model.

FIGS. 2A-2D are graphs showing the effects of SNALP-siRNAs on bodyweight in a Hep3B mouse model.

FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight ina Hep3B mouse model.

FIG. 4 is a graph showing the body weight in untreated control animals.

FIG. 5 is a graph showing the effects of control luciferase-SNALP siRNAson body weight in a Hep3B mouse model.

FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weightin a Hep3B mouse model.

FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDHlevels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFPlevels as measured by serum ELISA in a Hep3B mouse model.

FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDHlevels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 9 is a graph showing the effects of SNALP-siRNAs on human KSPlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGFlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGFlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on humanGAPDH levels and serum AFP levels in a Hep3B mouse model.

FIGS. 12A-12C are graphs showing the effects of SNALP-siRNAs on tumorKSP, VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 13A and FIG. 13B are graphs showing the effects of SNALP-siRNAs onsurvival in mice with hepatic tumors. Treatment was started at 18 days(FIG. 13A) and 26 days (FIG. 13B) after tumor cell seeding.

FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alphafetoprotein (AFP) levels.

FIGS. 15A and 15B are images of H&E stained sections in tumor bearinganimals (three weeks after Hep3B cell implantation) were administered 2mg/kg SNALP-VSP (A) or 2 mg/kg SNALP-Luc (B). Twenty four hours later,tumor bearing liver lobes were processed for histological analysis.Arrows indicate mono asters.

FIG. 16 is a flow diagram illustrating the manufacturing process ofALN-VSPDS01.

FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image ofALN-VSP02.

FIG. 18 is a flow diagram illustrating the manufacturing process ofALN-VSP02.

FIG. 19 is a graph illustrating the effects on survival ofadministration SNALP formulated siRNA and Sorafenib.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for inhibiting theexpression of the Eg5 gene and VEGF gene in a cell or mammal using thedsRNAs. The dsRNAs are preferably packaged in a stable nucleic acidparticle (SNALP). The invention also provides compositions and methodsfor treating pathological conditions and diseases, such as liver cancer,in a mammal caused by the expression of the Eg5 gene and VEGF genes. ThedsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi).

The following detailed description discloses how to make and use thecompositions containing dsRNAs to inhibit the expression of the Eg5 geneand VEGF genes, respectively, as well as compositions and methods fortreating diseases and disorders caused by the expression of these genes,such as cancer. The pharmaceutical compositions featured in theinvention include a dsRNA having an antisense strand comprising a regionof complementarity which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an RNA transcript of the Eg5 gene,together with a pharmaceutically acceptable carrier. The compositionsfeatured in the invention also include a dsRNA having an antisensestrand having a region of complementarity which is less than 30nucleotides in length, generally 19-24 nucleotides in length, and issubstantially complementary to at least part of an RNA transcript of theVEGF gene.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions containing the Eg5 and VEGF dsRNAs and a pharmaceuticallyacceptable carrier, methods of using the compositions to inhibitexpression of the Eg5 gene and the VEGF gene respectively, and methodsof using the pharmaceutical compositions to treat diseases caused byexpression of the Eg5 and VEGF genes.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. In another example, adenine and cytosine anywherein the oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences comprising such replacement moieties are embodiments of theinvention.

As used herein, “Eg5” refers to the human kinesin family member 11,which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIPS. Eg5sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeqID number:NM_(—)004523. The terms “Eg5” and “KSP” and “Eg5/KSP are usedinterchangeably

As used herein, VEGF, also known as vascular permeability factor, is anangiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein thatexists in at least three different isoforms. VEGF isoforms are expressedin endothelial cells. The VEGF gene contains 8 exons that express a189-amino acid protein isoform. A 165-amino acid isoform lacks theresidues encoded by exon 6, whereas a 121-amino acid isoform lacks theresidues encoded by exons 6 and 7. VEGF145 is an isoform predicted tocontain 145 amino acids and to lack exon 7. VEGF can act on endothelialcells by binding to an endothelial tyrosine kinase receptor, such asFlt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed inendothelial cells and is involved in endothelial cell differentiationand vasculogenesis. A third receptor, VEGFR-3, has been implicated inlymphogenesis.

The various isoforms have different biologic activities and clinicalimplications. For example, VEGF145 induces angiogenesis and like VEGF189(but unlike VEGF165) VEGF145 binds efficiently to the extracellularmatrix by a mechanism that is not dependent on extracellularmatrix-associated heparin sulfates. VEGF displays activity as anendothelial cell mitogen and chemoattractant in vitro and inducesvascular permeability and angiogenesis in vivo. VEGF is secreted by awide variety of cancer cell types and promotes the growth of tumors byinducing the development of tumor-associated vasculature Inhibition ofVEGF function has been shown to limit both the growth of primaryexperimental tumors as well as the incidence of metastases inimmunocompromised mice. Various dsRNAs directed to VEGF are described inco-pending U.S. Ser. No. 11/078,073 and 11/340,080, which are herebyincorporated by reference in their entirety.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the Eg5/KSP and/or VEGF gene, including mRNA that is a product of RNAprocessing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5′ UTR, anopen reading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a Eg5 mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aduplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA minus any overhangs that are present in the duplex. Inaddition to the duplex structure, a dsRNA may comprise one or morenucleotide overhangs. In general, the majority of nucleotides of eachstrand are ribonucleotides, but as described in detail herein, each orboth strands can also include at least one non-ribonucleotide, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. In some embodiments the dsRNA can have anucleotide overhang at one end of the duplex and a blunt end at theother end.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally the most tolerated mismatches are in theterminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA agent or aplasmid from which an iRNA agent is transcribed.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to the Eg5 and/or VEGF gene, herein refer to the at leastpartial suppression of the expression of the Eg5 gene, as manifested bya reduction of the amount of Eg5 mRNA and/or VEGF mRNA which may beisolated from a first cell or group of cells in which the Eg5 and/orVEGF gene is transcribed and which has or have been treated such thatthe expression of the Eg5 and/or VEGF gene is inhibited, as compared toa second cell or group of cells substantially identical to the firstcell or group of cells but which has or have not been so treated(control cells). The degree of inhibition is usually expressed in termsof

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Eg5 and/or VEGFgene expression, e.g. the amount of protein encoded by the Eg5 and/orVEGF gene which is produced by a cell, or the number of cells displayinga certain phenotype, e.g. apoptosis. In principle, target gene silencingcan be determined in any cell expressing the target, eitherconstitutively or by genomic engineering, and by any appropriate assay.However, when a reference is needed in order to determine whether agiven dsRNA inhibits the expression of the Eg5 gene by a certain degreeand therefore is encompassed by the instant invention, the assayprovided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the Eg5 gene (or VEGFgene) is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, or 50% by administration of the double-strandedoligonucleotide of the invention. In some embodiments, the Eg5 and/orVEGF gene is suppressed by at least about 60%, 70%, or 80% byadministration of the double-stranded oligonucleotide of the invention.In other embodiments, the Eg5 and/or VEGF gene is suppressed by at leastabout 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. The Tables and Example below providesvalues for inhibition of expression using various Eg5 and/or VEGF dsRNAmolecules at various concentrations.

As used herein in the context of Eg5 expression (or VEGF expression),the terms “treat”, “treatment”, and the like, refer to relief from oralleviation of pathological processes mediated by Eg5 and/or VEGFexpression. In the context of the present invention insofar as itrelates to any of the other conditions recited herein below (other thanpathological processes mediated by Eg5 and/or VEGF expression), theterms “treat”, “treatment”, and the like mean to relieve or alleviate atleast one symptom associated with such condition, or to slow or reversethe progression of such condition, such as the slowing and progressionof hepatic carcinoma.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by Eg5 and/or VEGF expression or anovert symptom of pathological processes mediated by Eg5 and/or VEGFexpression. The specific amount that is therapeutically effective can bereadily determined by ordinary medical practitioner, and may varydepending on factors known in the art, such as, e.g. the type ofpathological processes mediated by Eg5 and/or VEGF expression, thepatient's history and age, the stage of pathological processes mediatedby Eg5 and/or VEGF expression, and the administration of otheranti-pathological processes mediated by Eg5 and/or VEGF expressionagents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. As described in more detailbelow, such carriers include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The term specifically excludes cell culture medium. For drugsadministered orally, pharmaceutically acceptable carriers include, butare not limited to pharmaceutically acceptable excipients such as inertdiluents, disintegrating agents, binding agents, lubricating agents,sweetening agents, flavoring agents, coloring agents and preservatives.Suitable inert diluents include sodium and calcium carbonate, sodium andcalcium phosphate, and lactose, while corn starch and alginic acid aresuitable disintegrating agents. Binding agents may include starch andgelatin, while the lubricating agent, if present, will generally bemagnesium stearate, stearic acid or talc. If desired, the tablets may becoated with a material such as glyceryl monostearate or glyceryldistearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)

As described in more detail below, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of the Eg5 and/or VEGF gene in a cell or mammal, wherein thedsRNA comprises an antisense strand comprising a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of the Eg5 and/or VEGF gene, and wherein theregion of complementarity is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and wherein said dsRNA, uponcontact with a cell expressing said Eg5 and/or VEGF gene, inhibits theexpression of said Eg5 and/or VEGF gene.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

The dsRNA comprises two strands that are sufficiently complementary tohybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the Eg5 and/or VEGF gene, the other strand (the sensestrand) comprises a region which is complementary to the antisensestrand, such that the two strands hybridize and form a duplex structurewhen combined under suitable conditions. Generally, the duplex structureis between 15 and 30, more generally between 18 and 25, yet moregenerally between 19 and 24, and most generally between 19 and 21 basepairs in length. In other embodiments the duplex structure is 25-30 basepairs in length.

In one embodiment the duplex is 19 base pairs in length. In anotherembodiment the duplex is 21 base pairs in length. When two differentsiRNAs are used in combination, the duplex lengths can be identical orcan differ. For example, a composition can include a first dsRNAtargeted to Eg5 with a duplex length of 19 base pairs and a second dsRNAtargeted to VEGF with a duplex length of 21 base pairs.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 19 and 21 nucleotides inlength. In other embodiments the region of complementarity is 25-30nucleotides in length.

In one embodiment the region of complementarity is 19 nucleotides inlength. In another embodiment the region of complementarity is 21nucleotides in length. When two different siRNAs are used incombination, the region of complementarity can be identical or candiffer. For example, a composition can include a first dsRNA targeted toEg5 with a region of complementarity of 19 nucleotides and a seconddsRNA targeted to VEGF with a region of complementarity of 21nucleotides.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides inlength. In other embodiments, each is strand is 25-30 base pairs inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ. Forexample, a composition can include a dsRNA targeted to Eg5 with a sensestrand of 21 nucleotides and an antisense strand of 21 nucleotides, anda second dsRNA targeted to VEGF with a sense strand of 21 nucleotidesand an antisense strand of 23 nucleotides.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

As described in more detail herein, the composition of the inventionincludes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF.The first and second dsRNA can have the same overhang architecture,e.g., number of nucleotide overhangs on each strand, or each dsRNA canhave a different architecture. In one embodiment, the first dsRNAtargeting Eg5 includes a 2 nucleotide overhang at the 3′ end of eachstrand and the second dsRNA targeting VEGF includes a 2 nucleotideoverhang on the 3′ end of the antisense strand and a blunt end at the 5′end of the antisense strand (e.g., the 3′ end of the sense strand).

In one embodiment, the Eg5 gene targeted by the dsRNA of the inventionis the human Eg5 gene. In one embodiment, the antisense strand of thedsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of oneof the antisense sequences of Table 1-3. In specific embodiments, thefirst sequence of the dsRNA is selected from one of the sense strands ofTables 1-3 and the second sequence is selected from the group consistingof the antisense sequences of Tables 1-3. Alternative antisense agentsthat target elsewhere in the target sequence provided in Tables 1-3 canreadily be determined using the target sequence and the flanking Eg5sequence. In some embodiments the dsRNA targeted to Eg5 will comprise atleast two nucleotide sequence selected from the groups of sequencesprovided in Tables 1-3. One of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of the Eg5 gene. As such, the dsRNA will comprises twooligonucleotides, wherein one oligonucleotide is described as the sensestrand in Tables 1-3 and the second oligonucleotide is described as theantisense strand in Tables 1-3

In embodiments using a second dsRNA targeting VEGF, such agents areexemplified in the Examples, Tables 4a and 4b, and in co-pending U.S.Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.In one embodiment the dsRNA targeting VEGF has an antisense strandcomplementary to at least 15 contiguous nucleotides of the VEGF targetsequences described in Table 4a. In other embodiments, the dsRNAtargeting VEGF comprises one of the antisense sequences of Table 4b, orone of the sense sequences of Table 4b, or comprises one of the duplexes(sense and antisense strands) of Table 4b.

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1-3, the dsRNAs of theinvention can comprise at least one strand of a length of minimally 21nt. It can be reasonably expected that shorter dsRNAs comprising one ofthe sequences of Tables 1-3 minus only a few nucleotides on one or bothends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs comprising a partial sequence of at least 15, 16,17, 18, 19, 20, or more contiguous nucleotides from one of the sequencesof Tables 1-3, and differing in their ability to inhibit the expressionof the Eg5 gene in a FACS assay as described herein below by not morethan 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising thefull sequence, are contemplated by the invention. Further dsRNAs thatcleave within the target sequence provided in Tables 1-3 can readily bemade using the Eg5 sequence and the target sequence provided. AdditionaldsRNA targeting VEGF can be designed in a similar matter using thesequences disclosed in Tables 4a and 4b, the Examples and co-pendingU.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated byreference.

In addition, the RNAi agents provided in Tables 1-3 identify a site inthe Eg5 mRNA that is susceptible to RNAi based cleavage. As such thepresent invention further includes RNAi agents, e.g., dsRNA, that targetwithin the sequence targeted by one of the agents of the presentinvention. As used herein a second RNAi agent is said to target withinthe sequence of a first RNAi agent if the second RNAi agent cleaves themessage anywhere within the mRNA that is complementary to the antisensestrand of the first RNAi agent. Such a second agent will generallyconsist of at least 15 contiguous nucleotides from one of the sequencesprovided in Tables 1-3 coupled to additional nucleotide sequences takenfrom the region contiguous to the selected sequence in the Eg5 gene. Forexample, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6nucleotides from the target Eg5 gene produces a single strand agent of21 nucleotides that is based on one of the sequences provided in Tables1-3. Additional RNAi agents, e.g., dsRNA, targeting VEGF can be designedin a similar matter using the sequences disclosed in Tables 4a and 4b,the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080,herein incorporated by reference.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the Eg5 gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the Eg5 gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the Eg5 gene is important,especially if the particular region of complementarity in the Eg5 geneis known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability.

The nucleic acids of the invention may be synthesized and/or modified bymethods well established in the art, such as those described in “Currentprotocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.),John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosine's, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of an dsRNA compound. These dsRNAs typically contain atleast one region wherein the dsRNA is modified so as to confer upon thedsRNA increased resistance to nuclease degradation, increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

In some cases, a ligand can be multifunctional and/or a dsRNA can beconjugated to more than one ligand. For example, the dsRNA can beconjugated to one ligand for improved uptake and to a second ligand forimproved release.

Vector Encoded RNAi Agents

In another aspect of the invention, Eg5 and VEGF specific dsRNAmolecules that are expressed from transcription units inserted into DNAor RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10;Skillern, A., et al., International PCT Publication No. WO 00/22113,Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S.Pat. No. 6,054,299). These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl.Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector having, for example, either the U6 or H1RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g., the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell. dsRNAexpression DNA plasmids are typically transfected into target cells as acomplex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single EG5 gene (or VEGF gene) or multiple Eg5 genes (or VEGFgenes) over a period of a week or more are also contemplated by theinvention. Successful introduction of the vectors of the invention intohost cells can be monitored using various known methods. For example,transient transfection. can be signaled with a reporter, such as afluorescent marker, such as Green Fluorescent Protein (GFP). Stabletransfection of ex vivo cells can be ensured using markers that providethe transfected cell with resistance to specific environmental factors(e.g., antibiotics and drugs), such as hygromycin B resistance.

The Eg5 specific dsRNA molecules and VEGF specific dsRNA molecules canalso be inserted into vectors and used as gene therapy vectors for humanpatients. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier and methods of administering the same. Thepharmaceutical composition containing the dsRNA is useful for treating adisease or disorder associated with the expression or activity of aEg5/KSP and/or VEGF gene, such as pathological processes mediated byEg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceuticalcompositions are formulated based on the mode of delivery.

Dosage

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of EG5/KSP and/or VEGF genes.In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 1 to 50 mg per kilogram body weight per day.For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg,0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

The pharmaceutical composition can be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day. The effect of a single dose onEG5/KSP AND/OR VEGF levels is long lasting, such that subsequent dosesare administered at not more than 7 day intervals, or at not more than1, 2, 3, or 4 week intervals.

In some embodiments the dsRNA is administered using continuous infusionor delivery through a controlled release formulation. In that case, thedsRNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of thedsRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by EG5/KSP AND/OR VEGF expression. Such models are used for invivo testing of dsRNA, as well as for determining a therapeuticallyeffective dose. A suitable mouse model is, for example, a mousecontaining a plasmid expressing human EG5/KSP AND/OR VEGF. Anothersuitable mouse model is a transgenic mouse carrying a transgene thatexpresses human EG5/KSP AND/OR VEGF.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby target gene expression. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Administration

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, and subdermal,oral or parenteral, e.g., subcutaneous.

Typically, when treating a mammal with hyperlipidemia, the dsRNAmolecules are administered systemically via parental means. Parenteraladministration includes intravenous, intra-arterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intraparenchymal, intrathecal or intraventricular, administration.For example, dsRNAs, conjugated or unconjugate or formulated with orwithout liposomes, can be administered intravenously to a patient. Forsuch, a dsRNA molecule can be formulated into compositions such assterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions in liquid or solid oilbases. Such solutions also can contain buffers, diluents, and othersuitable additives. For parenteral, intrathecal, or intraventricularadministration, a dsRNA molecule can be formulated into compositionssuch as sterile aqueous solutions, which also can contain buffers,diluents, and other suitable additives (e.g., penetration enhancers,carrier compounds, and other pharmaceutically acceptable carriers).Formulations are described in more detail herein.

The dsRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Formulations

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. In one aspectare formulations that target the liver when treating hepatic disorderssuch as hyperlipidemia.

In addition, dsRNA that target the EG5/KSP AND/OR VEGF gene can beformulated into compositions containing the dsRNA admixed, encapsulated,conjugated, or otherwise associated with other molecules, molecularstructures, or mixtures of nucleic acids. For example, a compositioncontaining one or more dsRNA agents that target the Eg5/KSP and/or VEGFgene can contain other therapeutic agents such as other cancertherapeutics or one or more dsRNA compounds that target non-EG5/KSPAND/OR VEGF genes.

Oral, Parenteral, Topical, and Biologic Formulations

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, U.S. patent publication. No. 20030027780, and U.S. Pat.No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Suitable topical formulationsinclude those in which the dsRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). DsRNAs featured in the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, dsRNAs may be complexedto lipids, in particular to cationic lipids. Suitable fatty acids andesters include but are not limited to arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference. In addition, dsRNA molecules can beadministered to a mammal as biologic or abiologic means as described in,for example, U.S. Pat. No. 6,271,359. Abiologic delivery can beaccomplished by a variety of methods including, without limitation, (1)loading liposomes with a dsRNA acid molecule provided herein and (2)complexing a dsRNA molecule with lipids or liposomes to form nucleicacid-lipid or nucleic acid-liposome complexes. The liposome can becomposed of cationic and neutral lipids commonly used to transfect cellsin vitro. Cationic lipids can complex (e.g., charge-associate) withnegatively charged nucleic acids to form liposomes. Examples of cationicliposomes include, without limitation, lipofectin, lipofectamine,lipofectace, and DOTAP. Procedures for forming liposomes are well knownin the art. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including Lipofectin™ (Invitrogen/Life Technologies,Carlsbad, Calif) and Effectene™ (Qiagen, Valencia, Calif). In addition,systemic delivery methods can be optimized using commercially availablecationic lipids such as DDAB or DOTAP, each of which can be mixed with aneutral lipid such as DOPE or cholesterol. In some cases, liposomes suchas those described by Templeton et al. (Nature Biotechnology, 15:647-652 (1997)) can be used. In other embodiments, polycations such aspolyethyleneimine can be used to achieve delivery in vivo and ex vivo(Boletta et al., J. Am. Soc. Nephrol. 7: 1728 (1996)). Additionalinformation regarding the use of liposomes to deliver nucleic acids canbe found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 andMorrissey, D. et al. 2005. Nat. Biotechnol. 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including,without limitation, the use of viral vectors. For example, viral vectors(e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNAmolecules to liver cells. Standard molecular biology techniques can beused to introduce one or more of the dsRNAs provided herein into one ofthe many different viral vectors previously developed to deliver nucleicacid to cells. These resulting viral vectors can be used to deliver theone or more dsRNAs to cells by, for example, infection.

Characterization of Formulated dsRNAs

Formulations prepared by either the in-line mixing or extrusion-freemethod can be characterized in similar manners. For example,formulations are typically characterized by visual inspection. Theyshould be whitish translucent solutions free from aggregates orsediment. Particle size and particle size distribution oflipid-nanoparticles can be measured by light scattering using, forexample, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should beabout 20-300 nm, such as 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA can be incubated withan RNA-binding dye, such as Ribogreen (Molecular Probes) in the presenceor absence of a formulation disrupting surfactant, e.g., 0.5%Triton-X100. The total siRNA in the formulation can be determined by thesignal from the sample containing the surfactant, relative to a standardcurve. The entrapped fraction is determined by subtracting the “free”siRNA content (as measured by the signal in the absence of surfactant)from the total siRNA content. Percent entrapped siRNA is typically >85%.For SNALP formulation, the particle size is at least 30 nm, at least 40nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, atleast 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Thesuitable range is typically about at least 50 nm to about at least 110nm, about at least 60 nm to about at least 100 nm, or about at least 80nm to about at least 90 nm.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245) Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

SNALPs

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, orother nucleic acid-lipid particle. As used herein, the term “SNALP”refers to a stable nucleic acid-lipid particle, including SPLP. As usedherein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 to about90 nm, and are substantially nontoxic. In addition, the nucleic acidswhen present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof, or a mixture thereof. The cationic lipid may comprisefrom about 20 mol % to about 50 mol % or about 40 mol % of the totallipid present in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98·4HCl (MW 1487) (Formula 1),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

dsRNAs of the present invention can be formulated in a pharmaceuticallyacceptable carrier or diluent. A “pharmaceutically acceptable carrier”(also referred to herein as an “excipient”) is a pharmaceuticallyacceptable solvent, suspending agent, or any other pharmacologicallyinert vehicle. Pharmaceutically acceptable carriers can be liquid orsolid, and can be selected with the planned manner of administration inmind so as to provide for the desired bulk, consistency, and otherpertinent transport and chemical properties. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation:water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra-circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it isco-administered with polyinosinic acid, dextran sulfate, polycytidicacid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyaoet al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA &Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination Therapy

In one aspect, a composition of the invention can be used in combinationtherapy. The term “combination therapy” includes the administration ofthe subject compounds in further combination with other biologicallyactive ingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g., HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 andAEE788. Such combinations may enhance therapeutic efficacy over efficacyachieved by any of the agents alone and may prevent or delay theappearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA). In certain preferred embodiments, the compounds of theinvention are administered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (amcinonide, betamethasone,betamethasone dipropionate, betamethasone valerate, budesonide,clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol17-propionate, cortisone, deflazacort, desoximetasone, diflucortolonevalerate, dexamethasone, dexamethasone sodium phosphate, desonide,furoate, fluocinonide, fluocinolone acetonide, halcinonide,hydrocortisone, hydrocortisone butyrate, hydrocortisone sodiumsuccinate, hydrocortisone valerate, methyl prednisolone, mometasone,prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, andhalobetasol proprionate); a 5HTi agonist, such as a triptan (e.g.sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; anNMDA modulator, such as a glycine antagonist; a sodium channel blocker(e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilizing antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor α; an antibody therapy, such as a monoclonal antibody therapy; anantiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or animmune system modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

The compounds of the invention can be co-administered with siRNA thattarget other genes. For example, a compound of the invention can beco-administered with an siRNA targeted to a c-Myc gene. In one example,AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myctargeted siRNAs are disclosed in U.S. patent application Ser. No.12/373,039 which is herein incorporated by reference.

Methods for Treating Diseases Caused by Expression of the Eg5 and VEGFGenes

The invention relates in particular to the use of a compositioncontaining at least two dsRNAs, one targeting an Eg5 gene, and onetargeting a VEGF gene, for the treatment of a cancer, such as livercancer, e.g., for inhibiting tumor growth and tumor metastasis. Forexample, a composition, such as pharmaceutical composition, may be usedfor the treatment of solid tumors, like intrahepatic tumors such as mayoccur in cancers of the liver. A composition containing a dsRNAtargeting Eg5 and a dsRNA targeting VEGF may also be used to treat othertumors and cancers, such as breast cancer, lung cancer, head and neckcancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer,esophagus cancer, gastrointestinal cancer, glioma, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma and for thetreatment of skin cancer, like melanoma, for the treatment of lymphomasand blood cancer. The invention further relates to the use of acomposition containing an Eg5 dsRNA and a VEGF dsRNA for inhibitingaccumulation of ascites fluid and pleural effusion in different types ofcancer, e.g., liver cancer, breast cancer, lung cancer, head cancer,neck cancer, brain cancer, abdominal cancer, colon cancer, colorectalcancer, esophagus cancer, gastrointestinal cancer, glioma, tonguecancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer,prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skincancer, melanoma, lymphomas and blood cancer. Owing to the inhibitoryeffects on Eg5 and VEGF expression, a composition according to theinvention or a pharmaceutical composition prepared therefrom can enhancethe quality of life.

In one embodiment, a patient having a tumor associated with AFPexpression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, istreated. In certain embodiments, the patient has a malignant teratoma,an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, ahepatoblastoma, a heptocellular carcinoma, testicular cancer or ovariancancer.

The invention furthermore relates to the use of a dsRNA or apharmaceutical composition thereof, e.g., for treating cancer or forpreventing tumor metastasis, in combination with other pharmaceuticalsand/or other therapeutic methods, e.g., with known pharmaceuticalsand/or known therapeutic methods, such as, for example, those which arecurrently employed for treating cancer and/or for preventing tumormetastasis. Preference is given to a combination with radiation therapyand chemotherapeutic agents, such as cisplatin, cyclophosphamide,5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAiagent, in combination with another anti-cancer chemotherapeutic agent,such as any conventional chemotherapeutic agent. The combination of aspecific binding agent with such other agents can potentiate thechemotherapeutic protocol. Numerous chemotherapeutic protocols willpresent themselves in the mind of the skilled practitioner as beingcapable of incorporation into the method of the invention. Anychemotherapeutic agent can be used, including alkylating agents,antimetabolites, hormones and antagonists, radioisotopes, as well asnatural products. For example, the compound of the invention can beadministered with antibiotics such as doxorubicin and otheranthracycline analogs, nitrogen mustards such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinoma of thebreast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the compound can be administered inconjunction with leuprolide or goserelin (synthetic peptide analogs ofLH-RH). Other antineoplastic protocols include the use of a tetracyclinecompound with another treatment modality, e.g., surgery, radiation,etc., also referred to herein as “adjunct antineoplastic modalities.”Thus, the method of the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

Methods for Inhibiting Expression of the Eg5 Gene and the VEGF Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the Eg5 gene and the VEGF gene in a mammal. The methodincludes administering a composition featured in the invention to themammal such that expression of the target Eg5 gene and the target VEGFgene is silenced.

In one embodiment, a method for inhibiting Eg5 gene expression and VEGFgene expression includes administering a composition containing twodifferent dsRNA molecules, one having a nucleotide sequence that iscomplementary to at least a part of an RNA transcript of the Eg5 geneand the other having a nucleotide sequence that is complementary to atleast a part of an RNA transcript of the VEGF gene of the mammal to betreated. When the organism to be treated is a mammal such as a human,the composition may be administered by any means known in the artincluding, but not limited to oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),nasal, rectal, and topical (including buccal and sublingual)administration. In preferred embodiments, the compositions areadministered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

For screening of dsRNA, single-stranded RNAs were produced by solidphase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) andcontrolled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg,Germany) as solid support. RNA and RNA containing 2′-O-methylnucleotides were generated by solid phase synthesis employing thecorresponding phosphoramidites and 2′-O-methyl phosphoramidites,respectively (Proligo Biochemie GmbH, Hamburg, Germany). These buildingblocks were incorporated at selected sites within the sequence of theoligoribonucleotide chain using standard nucleoside phosphoramiditechemistry such as described in Current protocols in nucleic acidchemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., NewYork, N.Y., USA. Phosphorothioate linkages were introduced byreplacement of the iodine oxidizer solution with a solution of theBeaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%).Further ancillary reagents were obtained from Mallinckrodt Baker(Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

Conjugates

The following is a prophetic description of the synthesis of3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), anappropriately modified solid support was used for RNA synthesis. Themodified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stiffed at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

dsRNA Targeting the Eg5 Gene

Initial Screening Set

siRNA design was carried out to identify siRNAs targeting Eg5 (alsoknown as KIF11, HSKP, KNSL1 and TRIPS). Human mRNA sequences to Eg5,RefSeq ID number:NM_(—)004523, was used.

siRNA duplexes cross-reactive to human and mouse Eg5 were designed.Twenty-four duplexes were synthesized for screening. (Table 1a). Asecond screening set was defined with 266 siRNAs targeting human Eg5, aswell as its rhesus monkey ortholog (Table 2a). An expanded screening setwas selected with 328 siRNA targeting human Eg5, with no necessity tohit any Eg5 mRNA of other species (Table 3a).

The sequences for human and a partial rhesus Eg5 mRNAs were downloadedfrom NCBI Nucleotide database and the human sequence was further on usedas reference sequence (Human EG5:NM_(—)004523.2, 4908 bp, and RhesusEG5: XM_(—)001087644.1, 878 bp (only 5′ part of human EG5)

For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine:u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.

TABLE 1a Sequences of Eg5/KSP dsRNA duplexes position in human SEQ SEQSEQ Eg5/KSP ID sequence of 23mer target ID ID duplex sequence NO: siteNO: sense sequence (5′-3′) No: antisense sequence (5′-3′) name 385-4071244 ACCGAAGUGUUGUUUGUC 1 cGAAGuGuuGuuuGuccA 2 UUGGAcAAAcAAcACUUCGAL-DP- CAAUU ATsT TsT 6226 347-369 1245 UAUGGUGUUUGGAGCAUC 3uGGuGuuuGGAGcAucuA 4 GuAGAUGCUCcAAAcACcA AL-DP- UACUA cTsT TsT 62271078-1100 1246 AAUCUAAACUAACUAGAA 5 ucuAAAcuAAcuAGAAuc 6GGAUUCuAGUuAGUUuAGA AL-DP- UCCUC cTsT TsT 6228 1067-1089 1247UCCUUAUCGAGAAUCUAA 7 cuuAucGAGAAucuAAAc 8 AGUUuAGAUUCUCGAuAAG AL-DP-ACUAA uTsT TsT 6229 374-396 1248 GAUUGAUGUUUACCGAAG 9 uuGAuGuuuAccGAAGuG10 AcACUUCGGuAAAcAUcAA AL-DP- UGUUG uTsT TsT 6230 205-227 1249UGGUGAGAUGCAGACCAU 11 GuGAGAuGcAGAccAuuu 12 uAAAUGGUCUGcAUCUcAC AL-DP-UUAAU ATsT TsT 6231 1176-1198 1250 ACUCUGAGUACAUUGGAA 13ucuGAGuAcAuuGGAAuA 14 AuAUUCcAAUGuACUcAGA AL-DP- UAUGC uTsT TsT 6232386-408 1251 CCGAAGUGUUGUUUGUCC 15 GAAGuGuuGuuuGuccAA 16AUUGGAcAAAcAAcACUUC AL-DP- AAUUC uTsT TsT 6233 416-438 1252AGUUAUUAUGGGCUAUAA 17 uuAuuAuGGGcuAuAAuu 18 cAAUuAuAGCCcAuAAuAA AL-DP-UUGCA GTsT TsT 6234 485-507 1253 GGAAGGUGAAAGGUCACC 19AAGGuGAAAGGucAccuA 20 UuAGGUGACCUUUcACCUU AL-DP- UAAUG ATsT TsT 6235476-498 1254 UUUUACAAUGGAAGGUGA 21 uuAcAAuGGAAGGuGAAA 22CUUUcACCUUCcAUUGuAA AL-DP- AAGGU GTsT TsT 6236 486-508 1255GAAGGUGAAAGGUCACCU 23 AGGuGAAAGGucAccuAA 24 AUuAGGUGACCUUUcACCU AL-DP-AAUGA uTsT TsT 6237 487-509 1256 AAGGUGAAAGGUCACCUA 25GGuGAAAGGucAccuAAu 26 cAUuAGGUGACCUUUcACC AL-DP- AUGAA GTsT TsT 62381066-1088 1257 UUCCUUAUCGAGAAUCUA 27 ccuuAucGAGAAucuAAA 28GUUuAGAUUCUCGAuAAGG AL-DP- AACUA cTsT TsT 6239 1256-1278 1258AGCUCUUAUUAAGGAGUA 29 cucuuAuuAAGGAGuAuA 30 GuAuACUCCUuAAuAAGAG AL-DP-UACGG cTsT TsT 6240 2329-2351 1259 CAGAGAGAUUCUGUGCUU 31GAGAGAuucuGuGcuuuG 32 CcAAAGcAcAGAAUCUCUC AL-DP- UGGAG GTsT TsT 62411077-1099 1260 GAAUCUAAACUAACUAGA 33 AucuAAAcuAAcuAGAAu 34GAUUCuAGUuAGUUuAGAU AL-DP- AUCCU cTsT TsT 6242 1244-1266 1261ACUCACCAAAAAAGCUCU 35 ucAccAAAAAAGcucuuA 36 AuAAGAGCUUUUUUGGUGA AL-DP-UAUUA uTsT TsT 6243 637-659 1262 AAGAGCUUUUUGAUCUUC 37GAGcuuuuuGAucuucuu 38 uAAGAAGAUcAAAAAGCUC AL-DP- UUAAU ATsT TsT 62441117-1139 1263 GGCGUACAAGAACAUCUA 39 cGuAcAAGAAcAucuAuA 40UuAuAGAUGUUCUUGuACG AL-DP- UAAUU ATsT TsT 6245 373-395 1264AGAUUGAUGUUUACCGAA 41 AuuGAuGuuuAccGAAGu 42 cACUUCGGuAAAcAUcAAU AL-DP-GUGUU GTsT TsT 6246 1079-1101 1265 AUCUAAACUAACUAGAAU 43cuAAAcuAAcuAGAAucc 44 AGGAUUCuAGUuAGUUuAG AL-DP- CCUCC uTsT TsT 6247383-405 1266 UUACCGAAGUGUUGUUUG 45 AccGAAGuGuuGuuuGuc 46GGAcAAAcAAcACUUCGGU AL-DP- UCCAA cTsT TsT 6248 200-222 1267GGUGGUGGUGAGAUGCAG 47 uGGuGGuGAGAuGcAGAc 48 GGUCUGcAUCUcACcACcA AL-DP-ACCAU cTsT TsT 6249

TABLE 1b Analysis of Eg5/KSP ds duplexes single dose screen @ 25 nM [%SDs 2nd screen duplex residual (among name mRNA] quadruplicates)AL-DP-6226 23% 3% AL-DP-6227 69% 10% AL-DP-6228 33% 2% AL-DP-6229 2% 2%AL-DP-6230 66% 11% AL-DP-6231 17% 1% AL-DP-6232 9% 3% AL-DP-6233 24% 6%AL-DP-6234 91% 2% AL-DP-6235 112% 4% AL-DP-6236 69% 4% AL-DP-6237 42% 2%AL-DP-6238 45% 2% AL-DP-6239 2% 1% AL-DP-6240 48% 2% AL-DP-6241 41% 2%AL-DP-6242 8% 2% AL-DP-6243 7% 1% AL-DP-6244 6% 2% AL-DP-6245 12% 2%AL-DP-6246 28% 3% AL-DP-6247 71% 4% AL-DP-6248 5% 2% AL-DP-6249 28% 3%

TABLE 2a Sequences of Eg5/KSP dsRNA duplexes SEQ SEQ SEQ ID sequence of19-mer ID ID antisense sequence (5′- duplex NO: target site NO. sensesequence (5′-3′) NO. 3′) name 1268 CAUACUCUAGUCGUUCCCA 49cAuAcucuAGucGuucccATsT 50 UGGGAACGACuAGAGuAUGTsT AD-12072 1269AGCGCCCAUUCAAUAGUAG 51 AGcGcccAuucAAuAGuAGTsT 52 CuACuAUUGAAUGGGCGCUTsTAD-12073 1270 GGAAAGCUAGCGCCCAUUC 53 GGAAAGcuAGcGcccAuucTsT 54GAAUGGGCGCuAGCUUUCCTsT AD-12074 1271 GAAAGCUAGCGCCCAUUCA 55GAAAGcuAGcGcccAuucATsT 56 UGAAUGGGCGCuAGCUUUCTsT AD-12075 1272AGAAACUACGAUUGAUGGA 57 AGAAAcuAcGAuuGAuGGATsT 58 UCcAUcAAUCGuAGUUUCUTsTAD-12076 1273 UGUUCCUUAUCGAGAAUCU 59 uGuuccuuAucGAGAAucuTsT 60AGAUUCUCGAuAAGGAAcATsT AD-12077 1274 CAGAUUACCUCUGCGAGCC 61cAGAuuAccucuGcGAGccTsT 62 GGCUCGcAGAGGuAAUCUGTsT AD-12078 1275GCGCCCAUUCAAUAGUAGA 63 GcGcccAuucAAuAGuAGATsT 64 UCuACuAUUGAAUGGGCGCTsTAD-12079 1276 UUGCACUAUCUUUGCGUAU 65 uuGcAcuAucuuuGcGuAuTsT 66AuACGcAAAGAuAGUGcAATsT AD-12080 1277 CAGAGCGGAAAGCUAGCGC 67cAGAGcGGAAAGcuAGcGcTsT 68 GCGCuAGCUUUCCGCUCUGTsT AD-12081 1278AGACCUUAUUUGGUAAUCU 69 AGAccuuAuuuGGuAAucuTsT 70 AGAUuACcAAAuAAGGUCUTsTAD-12082 1279 AUUCUCUUGGAGGGCGUAC 71 AuucucuuGGAGGGcGuAcTsT 72GuACGCCCUCcAAGAGAAUTsT AD-12083 1280 GGCUGGUAUAAUUCCACGU 73GGcuGGuAuAAuuccAcGuTsT 74 ACGUGGAAUuAuACcAGCCTsT AD-12084 1281GCGGAAAGCUAGCGCCCAU 75 GcGGAAAGcuAGcGcccAuTsT 76 AUGGGCGCuAGCUUUCCGCTsTAD-12085 1282 UGCACUAUCUUUGCGUAUG 77 uGcAcuAucuuuGcGuAuGTsT 78cAuACGcAAAGAuAGUGcATsT AD-12086 1283 GUAUAAUUCCACGUACCCU 79GuAuAAuuccAcGuAcccuTsT 80 AGGGuACGUGGAAUuAuACTsT AD-12087 1284AGAAUCUAAACUAACUAGA 81 AGAAucuAAAcuAAcuAGATsT 82 UCuAGUuAGUUuAGAUUCUTsTAD-12088 1285 AGGAGCUGAAUAGGGUUAC 83 AGGAGcuGAAuAGGGuuAcTsT 84GuAACCCuAUUcAGCUCCUTsT AD-12089 1286 GAAGUACAUAAGACCUUAU 85GAAGuAcAuAAGAccuuAuTsT 86 AuAAGGUCUuAUGuACUUCTsT AD-12090 1287GACAGUGGCCGAUAAGAUA 87 GAcAGuGGccGAuAAGAuATsT 88 uAUCUuAUCGGCcACUGUCTsTAD-12091 1288 AAACCACUUAGUAGUGUCC 89 AAAccAcuuAGuAGuGuccTsT 90GGAcACuACuAAGUGGUUUTsT AD-12092 1289 UCCCUAGACUUCCCUAUUU 91ucccuAGAcuucccuAuuuTsT 92 AAAuAGGGAAGUCuAGGGATsT AD-12093 1290UAGACUUCCCUAUUUCGCU 93 uAGAcuucccuAuuucGcuTsT 94 AGCGAAAuAGGGAAGUCuATsTAD-12094 1291 GCGUCGCAGCCAAAUUCGU 95 GcGucGcAGccAAAuucGuTsT 96ACGAAUUUGGCUGCGACGCTsT AD-12095 1292 AGCUAGCGCCCAUUCAAUA 97AGcuAGcGcccAuucAAuATsT 98 uAUUGAAUGGGCGCuAGCUTsT AD-12096 1293GAAACUACGAUUGAUGGAG 99 GAAAcuAcGAuuGAuGGAGTsT 100 CUCcAUcAAUCGuAGUUUCTsTAD-12097 1294 CCGAUAAGAUAGAAGAUCA 101 ccGAuAAGAuAGAAGAucATsT 102UGAUCUUCuAUCUuAUCGGTsT AD-12098 1295 UAGCGCCCAUUCAAUAGUA 103uAGcGcccAuucAAuAGuATsT 104 uACuAUUGAAUGGGCGCuATsT AD-12099 1296UUUGCGUAUGGCCAAACUG 105 uuuGcGuAuGGccAAAcuGTsT 106cAGUUUGGCcAuACGcAAATsT AD-12100 1297 CACGUACCCUUCAUCAAAU 107cAcGuAcccuucAucAAAuTsT 108 AUUUGAUGAAGGGuACGUGTsT AD-12101 1298UCUUUGCGUAUGGCCAAAC 109 ucuuuGcGuAuGGccAAAcTsT 110GUUUGGCcAuACGcAAAGATsT AD-12102 1299 CCGAAGUGUUGUUUGUCCA 111ccGAAGuGuuGuuuGuccATsT 112 UGGAcAAAcAAcACUUCGGTsT AD-12103 1300AGAGCGGAAAGCUAGCGCC 113 AGAGcGGAAAGcuAGcGccTsT 114GGCGCuAGCUUUCCGCUCUTsT AD-12104 1301 GCUAGCGCCCAUUCAAUAG 115GcuAGcGcccAuucAAuAGTsT 116 CuAUUGAAUGGGCGCuAGCTsT AD-12105 1302AAGUUAGUGUACGAACUGG 117 AAGuuAGuGuAcGAAcuGGTsT 118CcAGUUCGuAcACuAACUUTsT AD-12106 1303 GUACGAACUGGAGGAUUGG 119GuAcGAAcuGGAGGAuuGGTsT 120 CcAAUCCUCcAGUUCGuACTsT AD-12107 1304ACGAACUGGAGGAUUGGCU 121 AcGAAcuGGAGGAuuGGcuTsT 122AGCcAAUCCUCcAGUUCGUTsT AD-12108 1305 AGAUUGAUGUUUACCGAAG 123AGAuuGAuGuuuAccGAAGTsT 124 CUUCGGuAAAcAUcAAUCUTsT AD-12109 1306UAUGGGCUAUAAUUGCACU 125 uAuGGGcuAuAAuuGcAcuTsT 126AGUGcAAUuAuAGCCcAuATsT AD-12110 1307 AUCUUUGCGUAUGGCCAAA 127AucuuuGcGuAuGGccAAATsT 128 UUUGGCcAuACGcAAAGAUTsT AD-12111 1308ACUCUAGUCGUUCCCACUC 129 AcucuAGucGuucccAcucTsT 130GAGUGGGAACGACuAGAGUTsT AD-12112 1309 AACUACGAUUGAUGGAGAA 131AAcuAcGAuuGAuGGAGAATsT 132 UUCUCcAUcAAUCGuAGUUTsT AD-12113 1310GAUAAGAGAGCUCGGGAAG 133 GAuAAGAGAGcucGGGAAGTsT 134CUUCCCGAGCUCUCUuAUCTsT AD-12114 1311 UCGAGAAUCUAAACUAACU 135ucGAGAAucuAAAcuAAcuTsT 136 AGUuAGUUuAGAUUCUCGATsT AD-12115 1312AACUAACUAGAAUCCUCCA 137 AAcuAAcuAGAAuccuccATsT 138UGGAGGAUUCuAGUuAGUUTsT AD-12116 1313 GGAUCGUAAGAAGGCAGUU 139GGAucGuAAGAAGGcAGuuTsT 140 AACUGCCUUCUuACGAUCCTsT AD-12117 1314AUCGUAAGAAGGCAGUUGA 141 AucGuAAGAAGGcAGuuGATsT 142UcAACUGCCUUCUuACGAUTsT AD-12118 1315 AGGCAGUUGACCAACACAA 143AGGcAGuuGAccAAcAcAATsT 144 UUGUGUUGGUcAACUGCCUTsT AD-12119 1316UGGCCGAUAAGAUAGAAGA 145 uGGccGAuAAGAuAGAAGATsT 146UCUUCuAUCUuAUCGGCcATsT AD-12120 1317 UCUAAGGAUAUAGUCAACA 147ucuAAGGAuAuAGucAAcATsT 148 UGUUGACuAuAUCCUuAGATsT AD-12121 1318ACUAAGCUUAAUUGCUUUC 149 AcuAAGcuuAAuuGcuuucTsT 150GAAAGcAAUuAAGCUuAGUTsT AD-12122 1319 GCCCAGAUCAACCUUUAAU 151GcccAGAucAAccuuuAAuTsT 152 AUuAAAGGUUGAUCUGGGCTsT AD-12123 1320UUAAUUUGGCAGAGCGGAA 153 uuAAuuuGGcAGAGcGGAATsT 154UUCCGCUCUGCcAAAUuAATsT AD-12124 1321 UUAUCGAGAAUCUAAACUA 155uuAucGAGAAucuAAAcuATsT 156 uAGUUuAGAUUCUCGAuAATsT AD-12125 1322CUAGCGCCCAUUCAAUAGU 157 cuAGcGcccAuucAAuAGuTsT 158ACuAUUGAAUGGGCGCuAGTsT AD-12126 1323 AAUAGUAGAAUGUGAUCCU 159AAuAGuAGAAuGuGAuccuTsT 160 AGGAUcAcAUUCuACuAUUTsT AD-12127 1324UACGAAAAGAAGUUAGUGU 161 uAcGAAAAGAAGuuAGuGuTsT 162AcACuAACUUCUUUUCGuATsT AD-12128 1325 AGAAGUUAGUGUACGAACU 163AGAAGuuAGuGuAcGAAcuTsT 164 AGUUCGuAcACuAACUUCUTsT AD-12129 1326ACUAAACAGAUUGAUGUUU 165 AcuAAAcAGAuuGAuGuuuTsT 166AAAcAUcAAUCUGUUuAGUTsT AD-12130 1327 CUUUGCGUAUGGCCAAACU 167cuuuGcGuAuGGccAAAcuTsT 168 AGUUUGGCcAuACGcAAAGTsT AD-12131 1328AAUGAAGAGUAUACCUGGG 169 AAuGAAGAGuAuAccuGGGTsT 170CCcAGGuAuACUCUUcAUUTsT AD-12132 1329 AUAAUUCCACGUACCCUUC 171AuAAuuccAcGuAcccuucTsT 172 GAAGGGuACGUGGAAUuAUTsT AD-12133 1330ACGUACCCUUCAUCAAAUU 173 AcGuAcccuucAucAAAuuTsT 174AAUUUGAUGAAGGGuACGUTsT AD-12134 1331 CGUACCCUUCAUCAAAUUU 175cGuAcccuucAucAAAuuuTsT 176 AAAUUUGAUGAAGGGuACGTsT AD-12135 1332GUACCCUUCAUCAAAUUUU 177 GuAcccuucAucAAAuuuuTsT 178AAAAUUUGAUGAAGGGuACTsT AD-12136 1333 AACUUACUGAUAAUGGUAC 179AAcuuAcuGAuAAuGGuAcTsT 180 GuACcAUuAUcAGuAAGUUTsT AD-12137 1334UUCAGUCAAAGUGUCUCUG 181 uucAGucAAAGuGucucuGTsT 182cAGAGAcACUUUGACUGAATsT AD-12138 1335 UUCUUAAUCCAUCAUCUGA 183uucuuAAuccAucAucuGATsT 184 UcAGAUGAUGGAUuAAGAATsT AD-12139 1336ACAGUACACAACAAGGAUG 185 AcAGuAcAcAAcAAGGAuGTsT 186cAUCCUUGUUGUGuACUGUTsT AD-12140 1337 AAGAAACUACGAUUGAUGG 187AAGAAAcuAcGAuuGAuGGTsT 188 CcAUcAAUCGuAGUUUCUUTsT AD-12141 1338AAACUACGAUUGAUGGAGA 189 AAAcuAcGAuuGAuGGAGATsT 190UCUCcAUcAAUCGuAGUUUTsT AD-12142 1339 UGGAGCUGUUGAUAAGAGA 191uGGAGcuGuuGAuAAGAGATsT 192 UCUCUuAUcAAcAGCUCcATsT AD-12143 1340CUAACUAGAAUCCUCCAGG 193 cuAAcuAGAAuccuccAGGTsT 194CCUGGAGGAUUCuAGUuAGTsT AD-12144 1341 GAAUAUGCUCAUAGAGCAA 195GAAuAuGcucAuAGAGcAATsT 196 UUGCUCuAUGAGcAuAUUCTsT AD-12145 1342AUGCUCAUAGAGCAAAGAA 197 AuGcucAuAGAGcAAAGAATsT 198UUCUUUGCUCuAUGAGcAUTsT AD-12146 1343 AAAAAUUGGUGCUGUUGAG 199AAAAAuuGGuGcuGuuGAGTsT 200 CUcAAcAGcACcAAUUUUUTsT AD-12147 1344GAGGAGCUGAAUAGGGUUA 201 GAGGAGcuGAAuAGGGuuATsT 202uAACCCuAUUcAGCUCCUCTsT AD-12148 1345 GGAGCUGAAUAGGGUUACA 203GGAGcuGAAuAGGGuuAcATsT 204 UGuAACCCuAUUcAGCUCCTsT AD-12149 1346GAGCUGAAUAGGGUUACAG 205 GAGcuGAAuAGGGuuAcAGTsT 206CUGuAACCCuAUUcAGCUCTsT AD-12150 1347 AGCUGAAUAGGGUUACAGA 207AGcuGAAuAGGGuuAcAGATsT 208 UCUGuAACCCuAUUcAGCUTsT AD-12151 1348GCUGAAUAGGGUUACAGAG 209 GcuGAAuAGGGuuAcAGAGTsT 210CUCUGuAACCCuAUUcAGCTsT AD-12152 1349 CCAAACUGGAUCGUAAGAA 211ccAAAcuGGAucGuAAGAATsT 212 UUCUuACGAUCcAGUUUGGTsT AD-12153 1350GAUCGUAAGAAGGCAGUUG 213 GAucGuAAGAAGGcAGuuGTsT 214cAACUGCCUUCUuACGAUCTsT AD-12154 1351 ACCUUAUUUGGUAAUCUGC 215AccuuAuuuGGuAAucuGcTsT 216 GcAGAUuACcAAAuAAGGUTsT AD-12155 1352UUAGAUACCAUUACUACAG 217 uuAGAuAccAuuAcuAcAGTsT 218CUGuAGuAAUGGuAUCuAATsT AD-12156 1353 AUACCAUUACUACAGUAGC 219AuAccAuuAcuAcAGuAGcTsT 220 GCuACUGuAGuAAUGGuAUTsT AD-12157 1354UACUACAGUAGCACUUGGA 221 uAcuAcAGuAGcAcuuGGATsT 222UCcAAGUGCuACUGuAGuATsT AD-12158 1355 AAAGUAAAACUGUACUACA 223AAAGuAAAAcuGuAcuAcATsT 224 UGuAGuAcAGUUUuACUUUTsT AD-12159 1356CUCAAGACUGAUCUUCUAA 225 cucAAGAcuGAucuucuAATsT 226UuAGAAGAUcAGUCUUGAGTsT AD-12160 1357 UUGACAGUGGCCGAUAAGA 227uuGAcAGuGGccGAuAAGATsT 228 UCUuAUCGGCcACUGUcAATsT AD-12161 1358UGACAGUGGCCGAUAAGAU 229 uGAcAGuGGccGAuAAGAuTsT 230AUCUuAUCGGCcACUGUcATsT AD-12162 1359 GCAAUGUGGAAACCUAACU 231GcAAuGuGGAAAccuAAcuTsT 232 AGUuAGGUUUCcAcAUUGCTsT AD-12163 1360CCACUUAGUAGUGUCCAGG 233 ccAcuuAGuAGuGuccAGGTsT 234CCUGGAcACuACuAAGUGGTsT AD-12164 1361 AGAAGGUACAAAAUUGGUU 235AGAAGGuAcAAAAuuGGuuTsT 236 AACcAAUUUUGuACCUUCUTsT AD-12165 1362UGGUUUGACUAAGCUUAAU 237 uGGuuuGAcuAAGcuuAAuTsT 238AUuAAGCUuAGUcAAACcATsT AD-12166 1363 GGUUUGACUAAGCUUAAUU 239GGuuuGAcuAAGcuuAAuuTsT 240 AAUuAAGCUuAGUcAAACCTsT AD-12167 1364UCUAAGUCAAGAGCCAUCU 241 ucuAAGucAAGAGccAucuTsT 242AGAUGGCUCUUGACUuAGATsT AD-12168 1365 UCAUCCCUAUAGUUCACUU 243ucAucccuAuAGuucAcuuTsT 244 AAGUGAACuAuAGGGAUGATsT AD-12169 1366CAUCCCUAUAGUUCACUUU 245 cAucccuAuAGuucAcuuuTsT 246AAAGUGAACuAuAGGGAUGTsT AD-12170 1367 CCCUAGACUUCCCUAUUUC 247cccuAGAcuucccuAuuucTsT 248 GAAAuAGGGAAGUCuAGGGTsT AD-12171 1368AGACUUCCCUAUUUCGCUU 249 AGAcuucccuAuuucGcuuTsT 250AAGCGAAAuAGGGAAGUCUTsT AD-12172 1369 UCACCAAACCAUUUGUAGA 251ucAccAAAccAuuuGuAGATsT 252 UCuAcAAAUGGUUUGGUGATsT AD-12173 1370UCCUUUAAGAGGCCUAACU 253 uccuuuAAGAGGccuAAcuTsT 254AGUuAGGCCUCUuAAAGGATsT AD-12174 1371 UUUAAGAGGCCUAACUCAU 255uuuAAGAGGccuAAcucAuTsT 256 AUGAGUuAGGCCUCUuAAATsT AD-12175 1372UUAAGAGGCCUAACUCAUU 257 uuAAGAGGccuAAcucAuuTsT 258AAUGAGUuAGGCCUCUuAATsT AD-12176 1373 GGCCUAACUCAUUCACCCU 259GGccuAAcucAuucAcccuTsT 260 AGGGUGAAUGAGUuAGGCCTsT AD-12177 1374UGGUAUUUUUGAUCUGGCA 261 uGGuAuuuuuGAucuGGcATsT 262UGCcAGAUcAAAAAuACcATsT AD-12178 1375 AGUUUAGUGUGUAAAGUUU 263AGuuuAGuGuGuAAAGuuuTsT 264 AAACUUuAcAcACuAAACUTsT AD-12179 1376GCCAAAUUCGUCUGCGAAG 265 GccAAAuucGucuGcGAAGTsT 266CUUCGcAGACGAAUUUGGCTsT AD-12180 1377 AAUUCGUCUGCGAAGAAGA 267AAuucGucuGcGAAGAAGATsT 268 UCUUCUUCGcAGACGAAUUTsT AD-12181 1378UGAAAGGUCACCUAAUGAA 269 uGAAAGGucAccuAAuGAATsT 270UUcAUuAGGUGACCUUUcATsT AD-12182 1379 CAGACCAUUUAAUUUGGCA 271cAGAccAuuuAAuuuGGcATsT 272 UGCcAAAUuAAAUGGUCUGTsT AD-12183 1380AGACCAUUUAAUUUGGCAG 273 AGAccAuuuAAuuuGGcAGTsT 274CUGCcAAAUuAAAUGGUCUTsT AD-12184 1381 AGUUAUUAUGGGCUAUAAU 275AGuuAuuAuGGGcuAuAAuTsT 276 AUuAuAGCCcAuAAuAACUTsT AD-12185 1382GCUGGUAUAAUUCCACGUA 277 GcuGGuAuAAuuccAcGuATsT 278uACGUGGAAUuAuACcAGCTsT AD-12186 1383 AUUUAAUUUGGCAGAGCGG 279AuuuAAuuuGGcAGAGcGGTsT 280 CCGCUCUGCcAAAUuAAAUTsT AD-12187 1384UUUAAUUUGGCAGAGCGGA 281 uuuAAuuuGGcAGAGcGGATsT 282UCCGCUCUGCcAAAUuAAATsT AD-12188 1385 UUUGGCAGAGCGGAAAGCU 283uuuGGcAGAGcGGAAAGcuTsT 284 AGCUUUCCGCUCUGCcAAATsT AD-12189 1386UUUUACAAUGGAAGGUGAA 285 uuuuAcAAuGGAAGGuGAATsT 286UUcACCUUCcAUUGuAAAATsT AD-12190 1387 AAUGGAAGGUGAAAGGUCA 287AAuGGAAGGuGAAAGGucATsT 288 UGACCUUUcACCUUCcAUUTsT AD-12191 1388UGAGAUGCAGACCAUUUAA 289 uGAGAuGcAGAccAuuuAATsT 290UuAAAUGGUCUGcAUCUcATsT AD-12192 1389 UCGCAGCCAAAUUCGUCUG 291ucGcAGccAAAuucGucuGTsT 292 cAGACGAAUUUGGCUGCGATsT AD-12193 1390GGCUAUAAUUGCACUAUCU 293 GGcuAuAAuuGcAcuAucuTsT 294AGAuAGUGcAAUuAuAGCCTsT AD-12194 1391 AUUGACAGUGGCCGAUAAG 295AuuGAcAGuGGccGAuAAGTsT 296 CUuAUCGGCcACUGUcAAUTsT AD-12195 1392CUAGACUUCCCUAUUUCGC 297 cuAGAcuucccuAuuucGcTsT 298GCGAAAuAGGGAAGUCuAGTsT AD-12196 1393 ACUAUCUUUGCGUAUGGCC 299AcuAucuuuGcGuAuGGccTsT 300 GGCcAuACGcAAAGAuAGUTsT AD-12197 1394AUACUCUAGUCGUUCCCAC 301 AuAcucuAGucGuucccAcTsT 302GUGGGAACGACuAGAGuAUTsT AD-12198 1395 AAAGAAACUACGAUUGAUG 303AAAGAAAcuAcGAuuGAuGTsT 304 cAUcAAUCGuAGUUUCUUUTsT AD-12199 1396GCCUUGAUUUUUUGGCGGG 305 GccuuGAuuuuuuGGcGGGTsT 306CCCGCcAAAAAAUcAAGGCTsT AD-12200 1397 CGCCCAUUCAAUAGUAGAA 307cGcccAuucAAuAGuAGAATsT 308 UUCuACuAUUGAAUGGGCGTsT AD-12201 1398CCUUAUUUGGUAAUCUGCU 309 ccuuAuuuGGuAAucuGcuTsT 310AGcAGAUuACcAAAuAAGGTsT AD-12202 1399 AGAGACAAUUCCGGAUGUG 311AGAGAcAAuuccGGAuGuGTsT 312 cAcAUCCGGAAUUGUCUCUTsT AD-12203 1400UGACUUUGAUAGCUAAAUU 313 uGAcuuuGAuAGcuAAAuuTsT 314AAUUuAGCuAUcAAAGUcATsT AD-12204 1401 UGGCAGAGCGGAAAGCUAG 315uGGcAGAGcGGAAAGcuAGTsT 316 CuAGCUUUCCGCUCUGCcATsT AD-12205 1402GAGCGGAAAGCUAGCGCCC 317 GAGcGGAAAGcuAGcGcccTsT 318GGGCGCuAGCUUUCCGCUCTsT AD-12206 1403 AAAGAAGUUAGUGUACGAA 319AAAGAAGuuAGuGuAcGAATsT 320 UUCGuAcACuAACUUCUUUTsT AD-12207 1404AUUGCACUAUCUUUGCGUA 321 AuuGcAcuAucuuuGcGuATsT 322uACGCAAAGAuAGUGcAAUTsT AD-12208 1405 GGUAUAAUUCCACGUACCC 323GGuAuAAuuccAcGuAcccTsT 324 GGGuACGUGGAAUuAuACCTsT AD-12209 1406UACUCUAGUCGUUCCCACU 325 uAcucuAGucGuucccAcuTsT 326AGUGGGAACGACuAGAGuATsT AD-12210 1407 UAUGAAAGAAACUACGAUU 327uAuGAAAGAAAcuAcGAuuTsT 328 AAUCGuAGUUUCUUUcAuATsT AD-12211 1408AUGCUAGAAGUACAUAAGA 329 AuGcuAGAAGuAcAuAAGATsT 330UCUuAUGuACUUCuAGcAUTsT AD-12212 1409 AAGUACAUAAGACCUUAUU 331AAGuAcAuAAGAccuuAuuTsT 332 AAuAAGGUCUuAUGuACUUTsT AD-12213 1410ACAGCCUGAGCUGUUAAUG 333 AcAGccuGAGcuGuuAAuGTsT 334cAUuAAcAGCUcAGGCUGUTsT AD-12214 1411 AAAGAAGAGACAAUUCCGG 335AAAGAAGAGAcAAuuccGGTsT 336 CCGGAAUUGUCUCUUCUUUTsT AD-12215 1412CACACUGGAGAGGUCUAAA 337 cAcAcuGGAGAGGucuAAATsT 338UUuAGACCUCUCcAGUGUGTsT AD-12216 1413 CACUGGAGAGGUCUAAAGU 339cAcuGGAGAGGucuAAAGuTsT 340 ACUUuAGACCUCUCcAGUGTsT AD-12217 1414ACUGGAGAGGUCUAAAGUG 341 AcuGGAGAGGucuAAAGuGTsT 342cACUUuAGACCUCUCcAGUTsT AD-12218 1415 CGUCGCAGCCAAAUUCGUC 343cGucGcAGccAAAuucGucTsT 344 GACGAAUUUGGCUGCGACGTsT AD-12219 1416GAAGGCAGUUGACCAACAC 345 GAAGGcAGuuGAccAAcAcTsT 346GUGUUGGUcAACUGCCUUCTsT AD-12220 1417 CAUUCACCCUGACAGAGUU 347cAuucAcccuGAcAGAGuuTsT 348 AACUCUGUcAGGGUGAAUGTsT AD-12221 1418AAGAGGCCUAACUCAUUCA 349 AAGAGGccuAAcucAuucATsT 350UGAAUGAGUuAGGCCUCUUTsT AD-12222 1419 GAGACAAUUCCGGAUGUGG 351GAGAcAAuuccGGAuGuGGTsT 352 CcAcAUCCGGAAUUGUCUCTsT AD-12223 1420UUCCGGAUGUGGAUGUAGA 353 uuccGGAuGuGGAuGuAGATsT 354UCuAcAUCcAcAUccGGAATsT AD-12224 1421 AAGCUAGCGCCCAUUCAAU 355AAGcuAGcGcccAuucAAuTsT 356 AUUGAAUGGGCGCuAGCUUTsT AD-12225 1422GAAGUUAGUGUACGAACUG 357 GAAGuuAGuGuAcGAAcuGTsT 358cAGUUCGuAcACuAACUUCTsT AD-12226 1423 UAUAAUUCCACGUACCCUU 359uAuAAuuccAcGuAcccuuTsT 360 AAGGGuACGUGGAAUuAuATsT AD-12227 1424ACAGUGGCCGAUAAGAUAG 361 AcAGuGGccGAuAAGAuAGTsT 362CuAUCUuAUCGGCcACUGUTsT AD-12228 1425 UCUGUCAUCCCUAUAGUUC 363ucuGucAucccuAuAGuucTsT 364 GAACuAuAGGGAUGAcAGATsT AD-12229 1426UUCUUGCUAUGACUUGUGU 365 uucuuGcuAuGAcuuGuGuTsT 366AcAcAAGUcAuAGcAAGAATsT AD-12230 1427 GUAAGAAGGCAGUUGACCA 367GuAAGAAGGcAGuuGAccATsT 368 UGGUcAACUGCCUUCUuACTsT AD-12231 1428CAUUGACAGUGGCCGAUAA 369 cAuuGAcAGuGGccGAuAATsT 370UuAUCGGCcACUGUcAAUGTsT AD-12232 1429 AGAAACCACUUAGUAGUGU 371AGAAAccAcuuAGuAGuGuTsT 372 AcAcuAcuAAGUGGUUUCUTsT AD-12233 1430GGAUUGUUCAUCAAUUGGC 373 GGAuuGuucAucAAuuGGcTsT 374GCcAAUUGAUGAAcAAUCCTsT AD-12234 1431 UAAGAGGCCUAACUCAUUC 375uAAGAGGccuAAcucAuucTsT 376 GAAUGAGUuAGGCCUCUuATsT AD-12235 1432AGUUAGUGUACGAACUGGA 377 AGuuAGuGuAcGAAcuGGATsT 378UCcAGUUcGuAcACuAACUTsT AD-12236 1433 AGUACAUAAGACCUUAUUU 379AGuAcAuAAGAccuuAuuuTsT 380 AAAuAAGGUCUuAUGuACUTsT AD-12237 1434UGAGCCUUGUGUAUAGAUU 381 uGAGccuuGuGuAuAGAuuTsT 382AAUCuAuAcAcAAGGCUcATsT AD-12238 1435 CCUUUAAGAGGCCUAACUC 383ccuuuAAGAGGccuAAcucTsT 384 GAGUuAGGCCUCUuAAAGGTsT AD-12239 1436ACCACUUAGUAGUGUCCAG 385 AccAcuuAGuAGuGuccAGTsT 386CUGGAcAcuAcuAAGUGGUTsT AD-12240 1437 GAAACUUCCAAUUAUGUCU 387GAAAcuuccAAuuAuGucuTsT 388 AGAcAuAAUUGGAAGUUUCTsT AD-12241 1438UGCAUACUCUAGUCGUUCC 389 uGcAuAcucuAGucGuuccTsT 390GGAACGACuAGAGuAUGcATsT AD-12242 1439 AGAAGGCAGUUGACCAACA 391AGAAGGcAGuuGAccAAcATsT 392 UGUUGGUcAACUGCCUUCUTsT AD-12243 1440GUACAUAAGACCUUAUUUG 393 GuAcAuAAGAccuuAuuuGTST 394cAAAuAAGGUCUuAUGuAcTsT AD-12244 1441 UAUAAUUGCACUAUCUUUG 395uAuAAuuGcAcuAucuuuGTsT 396 cAAAGAuAGUGcAAUuAuATsT AD-12245 1442UCUCUGUUACAAUACAUAU 397 ucucuGuuAcAAuAcAuAuTsT 398AuAUGuAUUGuAAcAGAGATsT AD-12246 1443 UAUGCUCAUAGAGCAAAGA 399uAuGcucAuAGAGcAAAGATsT 400 UCUUUGCUCuAUGAGcAuATsT AD-12247 1444UGUUGUUUGUCCAAUUCUG 401 uGuuGuuuGuccAAuucuGTsT 402cAGAAUUGGAcAAAcAAcATST AD-12248 1445 ACUAACUAGAAUCCUCCAG 403AcuAAcuAGAAuccuccAGTsT 404 CUGGAGGAUUCuAGUuAGUTsT AD-12249 1446UGUGGUGUCUAUACUGAAA 405 uGuGGuGucuAuAcuGAAATsT 406UUUcAGuAuAGAcACcAcATsT AD-12250 1447 UAUUAUGGGAGACCACCCA 407uAuuAuGGGAGAccAcccATsT 408 UGGGUGGUCUCCcAuAAuATsT AD-12251 1448AAGGAUGAAGUCUAUCAAA 409 AAGGAuGAAGucuAucAAATsT 410UUUGAuAGAcUUcAUCCUUTsT AD-12252 1449 UUGAUAAGAGAGCUCGGGA 411uuGAuAAGAGAGcucGGGATsT 412 UCCCGAGCUCUCUuAUcAATsT AD-12253 1450AUGUUCCUUAUCGAGAAUC 413 AuGuuccuuAucGAGAAucTsT 414GAUUCUCGAuAAGGAAcAUTsT AD-12254 1451 GGAAUAUGCUCAUAGAGCA 415GGAAuAuGcucAuAGAGcATsT 416 UGCUCuAUGAGcAuAUUCCTsT AD-12255 1452CCAUUCCAAACUGGAUCGU 417 ccAuuccAAAcuGGAucGuTsT 418ACGAUCcAGUUUGGAAUGGTsT AD-12256 1453 GGCAGUUGACCAACACAAU 419GGcAGuuGAccAAcAcAAuTsT 420 AUUGUGUUGGUcAACUGCCTsT AD-12257 1454CAUGCUAGAAGUACAUAAG 421 cAuGcuAGAAGuAcAuAAGTsT 422CUuAUGuACUUCuAGcAUGTsT AD-12258 1455 CUAGAAGUACAUAAGACCU 423cuAGAAGuAcAuAAGAccuTsT 424 AGGUCUuAUGuACUUCuAGTsT AD-12259 1456UUGGAUCUCUCACAUCUAU 425 uuGGAucucucAcAucuAuTsT 426AuAGAUGUGAGAGAUCcAATsT AD-12260 1457 AACUGUGGUGUCUAUACUG 427AAcuGuGGuGucuAuAcuGTsT 428 cAGuAuAGAcACcAcAGUUTsT AD-12261 1458UCAUUGACAGUGGCCGAUA 429 ucAuuGAcAGuGGccGAuATsT 430uAUCGGCcACUGUcAAUGATsT AD-12262 1459 AUAAAGCAGACCCAUUCCC 431AuAAAGcAGAcccAuucccTsT 432 GGGAAUGGGUCUGCUUuAUTsT AD-12263 1460ACAGAAACCACUUAGUAGU 433 AcAGAAAccAcuuAGuAGuTsT 434AcuAcuAAGUGGUUUCUGUTsT AD-12264 1461 GAAACCACUUAGUAGUGUC 435GAAAccAcuuAGuAGuGucTsT 436 GAcACuACuAAGUGGUUUCTsT AD-12265 1462AAAUCUAAGGAUAUAGUCA 437 AAAucuAAGGAuAuAGucATsT 438UGAcuAuAUCCUuAGAUUUTsT AD-12266 1463 UUAUUUAUACCCAUCAACA 439uuAuuuAuAcccAucAAcATsT 440 UGUUGAUGGGuAuAAAuAATsT AD-12267 1464ACAGAGGCAUUAACACACU 441 AcAGAGGcAuuAAcAcAcuTsT 442AGUGUGUuAAUGCCUCUGUTsT AD-12268 1465 ACACACUGGAGAGGUCUAA 443AcAcAcuGGAGAGGucuAATsT 444 UuAGACCUCUCcAGUGUGUTsT AD-12269 1466ACACUGGAGAGGUCUAAAG 445 AcAcuGGAGAGGucuAAAGTsT 446CUUuAGACCUCUCcAGUGUTsT AD-12270 1467 CGAGCCCAGAUCAACCUUU 447cGAGcccAGAucAAccuuuTsT 448 AAAGGUUGAUCUGGGCUCGTsT AD-12271 1468UCCCUAUUUCGCUUUCUCC 449 ucccuAuuucGcuuucuccTsT 450GGAGAAAGCGAAAuAGGGATsT AD-12272 1469 UCUAAAAUCACUGUCAACA 451ucuAAAAucAcuGucAAcATsT 452 UGUUGAcAGUGAUUUuAGATsT AD-12273 1470AGCCAAAUUCGUCUGCGAA 453 AGccAAAuucGucuGcGAATsT 454UUCGcAGACGAAUUUGGCUTsT AD-12274 1471 CCCAUUCAAUAGUAGAAUG 455cccAuucAAuAGuAGAAuGTsT 456 cAUUCuACuAUUGAAUGGGTsT AD-12275 1472GAUGAAUGCAUACUCUAGU 457 GAuGAAuGcAuAcucuAGuTsT 458ACuAGAGuAUGcAUUcAUCTsT AD-12276 1473 CUCAUGUUCCUUAUCGAGA 459cucAuGuuccuuAucGAGATsT 460 UCUCGAuAAGGAAcAUGAGTsT AD-12277 1474GAGAAUCUAAACUAACUAG 461 GAGAAucuAAAcuAAcuAGTsT 462CuAGUuAGUUuAGAUUCUCTsT AD-12278 1475 UAGAAGUACAUAAGACCUU 463uAGAAGuAcAuAAGAccuuTsT 464 AAGGUCUuAUGuACUUCuATsT AD-12279 1476CAGCCUGAGCUGUUAAUGA 465 cAGccuGAGcuGuuAAucATsT 466UcAUuAAcAGCUcAGGCUGTsT AD-12280 1477 AAGAAGAGACAAUUCCGGA 467AAGAAGAGAcAAuuccGGATsT 468 UCCGGAAUUGUCUCUUCUUTsT AD-12281 1478UGCUGGUGUGGAUUGUUCA 469 uGcuGGuGuGGAuuGuucATsT 470UGAAcAAUCcAcACcAGcATsT AD-12282 1479 AAAUUCGUCUGCGAAGAAG 471AAAuucGucuGcGAAGAAGTsT 472 CUUCUUCGcAGACGAAUUUTsT AD-12283 1480UUUCUGGAAGUUGAGAUGU 473 uuucuGGAAGuuGAGAuGuTsT 474AcAUCUcAACUUCcAGAAATsT AD-12284 1481 UACUAAACAGAUUGAUGUU 475uAcuAAAcAGAuuGAuGuuTsT 476 AAcAUcAAUCUGUUuAGuATsT AD-12285 1482GAUUGAUGUUUACCGAAGU 477 GAuuGAuGuuuAccGAAGuTsT 478ACUUCGGuAAAcAUcAAUCTsT AD-12286 1483 GCACUAUCUUUGCGUAUGG 479GcAcuAucuuuGcGuAuGGTsT 480 CcAuACGcAAAGAuAGUGCTsT AD-12287 1484UGGUAUAAUUCCACGUACC 481 uGGuAuAAuuccAcGuAccTsT 482GGuACGUGGAAUuAuACcATsT AD-12288 1485 AGCAAGCUGCUUAACACAG 483AGcAAGcuGcuuAAcAcAGTsT 484 CUGUGUuAAGcAGCUUGCUTsT AD-12289 1486CAGAAACCACUUAGUAGUG 485 cAGAAAccAcuuAGuAGuGTsT 486cACuACuAAGUGGUUUCUGTsT AD-12290 1487 AACUUAUUGGAGGUUGUAA 487AAcuuAuuGGAGGuuGuAATsT 488 UuAcAACCUCcAAuAAGUUTsT AD-12291 1488CUGGAGAGGUCUAAAGUGG 489 cuGGAGAGGucuAAAGuGGTsT 490CcACUUuAGACCUCUCcAGTsT AD-12292 1489 AAAAAAGAUAUAAGGCAGU 491AAAAAAGAuAuAAGGcAGuTsT 492 ACUGCCUuAuAUCUUUUUUTsT AD-12293 1490GAAUUUUGAUAUCUACCCA 493 GAAuuuuGAuAucuAcccATsT 494UGGGuAGAuAUcAAAAUUCTsT AD-12294 1491 GUAUUUUUGAUCUGGCAAC 495GuAuuuuuGAucuGGcAAcTsT 496 GUUGCcAGAUcAAAAAuACTsT AD-12295 1492AGGAUCCCUUGGCUGGUAU 497 AGGAucccuuGGcuGGuAuTsT 498AuACcAGCcAAGGGAUCCUTsT AD-12296 1493 GGAUCCCUUGGCUGGUAUA 499GGAucccuuGGcuGGuAuATsT 500 uAuACcAGCcAAGGGAUCCTsT AD-12297 1494CAAUAGUAGAAUGUGAUCC 501 cAAuAGuAGAAuGuGAuccTsT 502GGAUcAcAUUCuACuAUUGTsT AD-12298 1495 GCUAUAAUUGCACUAUCUU 503GcuAuAAuuGcAcuAucuuTsT 504 AAGAuAGUGcAAUuAuAGcTsT AD-12299 1496UACCCUUCAUCAAAUUUUU 505 uAcccuucAucAAAuuuuuTsT 506AAAAAUUUGAUGAAGGGuATsT AD-12300 1497 AGAACAUAUUGAAUAAGCC 507AGAAcAuAuuGAAuAAGccTsT 508 GGCUuAUUcAAuAUGUUCUTsT AD-12301 1498AAAUUGGUGCUGUUGAGGA 509 AAAuuGGuGcuGuuGAGGATsT 510UCCUcAAcAGcACcAAUUUTsT AD-12302 1499 UGAAUAGGGUUACAGAGUU 511uGAAuAGGGuuAcAGAGuuTsT 512 AACUCUGuAACCCuAUUcATsT AD-12303 1500AAGAACUUGAAACCACUCA 513 AAGAAcuuGAAAccAcucATsT 514UGAGUGGUUUcAAGUUCUUTsT AD-12304 1501 AAUAAAGCAGACCCAUUCC 515AAuAAAGcAGAcccAuuccTsT 516 GGAAUGGGUCUGCUUuAUUTsT AD-12305 1502AUACCCAUCAACACUGGUA 517 AuAcccAucAAcAcuGGuATsT 518uACcAGUGUUGAUGGGuAUTsT AD-12306 1503 UGGAUUGUUCAUCAAUUGG 519uGGAuuGuucAucAAuuGGTsT 520 CcAAUUGAUGAAcAAUCcATsT AD-12307 1504UGGAGAGGUCUAAAGUGGA 521 uGGAGAGGucuAAAGuGGATsT 522UCcACUUuAGACCUCUCcATsT AD-12308 1505 GUCAUCCCUAUAGUUCACU 523GucAucccuAuAGuucAcuTsT 524 AGUGAACuAuAGGGAUGACTsT AD-12309 1506AUAAUGGCUAUAAUUUCUC 525 AuAAuGGcuAuAAuuucucTsT 526GAGAAAUuAuAGCcAUuAUTsT AD-12310 1507 AUCCCUUGGCUGGUAUAAU 527AucccuuGGcuGGuAuAAuTsT 528 AUuAuACcAGCcAAGGGAUTsT AD-12311 1508GGGCUAUAAUUGCACUAUC 529 GGGcuAuAAuuGcAcuAucTsT 530GAuAGUGcAAUuAuAGCCCTsT AD-12312 1509 GAUUCUCUUGGAGGGCGUA 531GAuucucuuGGAGGGcGuATsT 532 uACGCCCUCcAAGAGAAUCTsT AD-12313 1510GCAUCUCUCAAUCUUGAGG 533 GcAucucucAAucuuGAGGTsT 534CCUcAAGAUUGAGAGAUGCTsT AD-12314 1511 CAGCAGAAAUCUAAGGAUA 535cAGcAGAAAucuAAGGAuATsT 536 uAUCCUuAGAUUUCUGCUGTsT AD-12315 1512GUCAAGAGCCAUCUGUAGA 537 GucAAGAGccAucuGuAGATsT 538UCuAcAGAUGGCUCUUGACTsT AD-12316 1513 AAACAGAGGCAUUAACACA 539AAAcAGAGGcAuuAAcAcATsT 540 UGUGUuAAUGCCUCUGUUUTsT AD-12317 1514AGCCCAGAUCAACCUUUAA 541 AGcccAGAucAAccuuuAATsT 542UuAAAGGUUGAUCUGGGCUTsT AD-12318 1515 UAUUUUUGAUCUGGCAACC 543uAuuuuuGAucuGGcAAccTsT 544 GGUUGCcAGAUcAAAAAuATsT AD-12319 1516UGUUUGGAGCAUCUACUAA 545 uGuuuGGAGcAucuAcuAATsT 546UuAGuAGAUGCUCcAAAcATsT AD-12320 1517 GAAAUUACAGUACACAACA 547GAAAuuAcAGuAcAcAAcATsT 548 UGUUGUGuACUGuAAUUUCTsT AD-12321 1518ACUUGACCAGUGUAAAUCU 549 AcuuGAccAGuGuAAAucuTsT 550AGAUUuAcACUGGUcAAGUTsT AD-12322 1519 ACCAGUGUAAAUCUGACCU 551AccAGuGuAAAucuGAccuTsT 552 AGGUcAGAUUuAcACUGGUTsT AD-12323 1520AGAACAAUCAUUAGCAGCA 553 AGAAcAAucAuuAGcAGcATsT 554UGCUGCuAAUGAUUGUUCUTsT AD-12324 1521 CAAUGUGGAAACCUAACUG 555cAAuGuGGAAAccuAAcuGTsT 556 cAGUuAGGUUUCcAcAUUGTsT AD-12325 1522ACCAAGAAGGUACAAAAUU 557 AccAAGAAGGuAcAAAAuuTsT 558AAUUUUGuACCUUCUUGGUTsT AD-12326 1523 GGUACAAAAUUGGUUGAAG 559GGuAcAAAAuuGGuuGAAGTsT 560 CUUcAACcAAUUUUGuACCTsT AD-12327 1524GGUGUGGAUUGUUCAUCAA 561 GGuGuGGAuuGuucAucAATsT 562UUGAUGAAcAAUCcAcACCTsT AD-12328 1525 AGAGUUCACAAAAAGCCCA 563AGAGuucAcAAAAAGcccATsT 564 UGGGCUUUUUGUGAACUCUTsT AD-12329 1526UGAUAGCUAAAUUAAACCA 565 uGAuAGcuAAAuuAAAccATsT 566UGGUUuAAUUuAGCuAUcATsT AD-12330 1527 AAUAAGCCUGAACUGAAUC 567AAuAAGccuGAAGuGAAucTsT 568 GAUUcACUUcAGGCUuAUUTsT AD-12331 1528CAGUUGACCAACACAAUGC 569 cAGuuGAccAAcAcAAuGcTsT 570GcAUUGUGUUGGUcAACUGTsT AD-12332 1529 UGGUGUGGAUUGUUCAUCA 571uGGuGuGGAuuGuucAucATsT 572 UGAUGAAcAAUCcAcACcATsT AD-12333 1530AUUCACCCUGACACAGUUC 573 AuucAcccuGAcAGAGuucTsT 574GAACUCUGUcAcGGUGAAUTsT AD-12334 1531 UAAGACCUUAUUUGGUAAU 575uAAGAccuuAuuuGGuAAuTsT 576 AUuACcAAAuAAGGUCUuATsT AD-12335 1532AAGCAAUGUGGAAACCUAA 577 AAGcAAuGuGGAAAccuAATsT 578UuAGGUUUccAcAUUGCUUTsT AD-12336 1533 UCUGAAACUGGAUAUCCCA 579ucuGAAAcuGGAuAucccATsT 580 UGGGAuAUCcAGUUUcAGATsT AD-12337

TABLE 2b Analysis of Eg5/KSP dsRNA duplexes 1st single 2nd single dosedose 3rd screen @ screen @ single Eg5/KSP 50 nm [% SDs 1st screen 25 nM[% SDs 2nd screen dose SDs 3rd screen duplex resudual (among resudual(among screen (among name mRNA] quadruplicates) mRNA] quadruplicates) @25 nM quadruplicates) AD-12072 65% 2% 82% 5% AD-12073 84% 1% 61% 6%AD-12074 51% 3% 36% 9% AD-12075 56% 4% 36% 4% AD-12076 21% 4% 13% 3%AD-12077 11% 2% 6% 1% AD-12078 22% 3% 9% 2% AD-12079 22% 10% 15% 7%AD-12080 68% 4% 52% 13% AD-12081 34% 8% 35% 24% AD-12082 20% 2% 92% 5%AD-12083 85% 6% 63% 10% AD-12084 18% 6% 17% 4% AD-12085 13% 4% 12% 4%AD-12086 26% 5% 17% 3% AD-12087 95% 4% 80% 4% AD-12088 29% 6% 29% 2%AD-12089 69% 5% 64% 7% AD-12090 46% 15% 34% 5% AD-12091 16% 6% 17% 3%AD-12092 82% 26% 63% 5% AD-12093 84% 4% 70% 4% AD-12094 46% 3% 34% 1%AD-12095 14% 2% 13% 1% AD-12096 26% 11% 17% 1% AD-12097 23% 2% 21% 1%AD-12098 41% 14% 17% 3% AD-12099 57% 2% 48% 6% AD-12100 101% 11% 98% 8%AD-12101 46% 7% 32% 2% AD-12102 96% 17% 88% 18% AD-12103 19% 5% 20% 2%AD-12104 40% 8% 24% 2% AD-12105 39% 2% 36% 10% AD-12106 87% 6% 79% 19%AD-12107 29% 2% 32% 16% AD-12108 38% 4% 39% 8% AD-12109 49% 3% 44% 10%AD-12110 85% 5% 80% 14% AD-12111 64% 6% 71% 18% AD-12112 48% 4% 41% 5%AD-12113 13% 0% 14% 3% AD-12114 32% 6% 16% 4% AD-12115 8% 4% 7% 5%AD-12116 74% 5% 61% 7% AD-12117 21% 4% 20% 2% AD-12118 44% 4% 42% 6%AD-12119 37% 4% 24% 3% AD-12120 22% 2% 15% 4% AD-12121 32% 1% 22% 2%AD-12122 36% 16% 19% 5% AD-12123 28% 1% 16% AD-12124 28% 2% 16% AD-1212515% 1% 14% AD-12126 51% 22% 27% AD-12127 54% 4% 42% 9% AD-12128 29% 1%20% 2% AD-12129 22% 3% 19% 3% AD-12130 53% 6% 42% 7% AD-12131 28% 5% 22%3% AD-12132 88% 2% 90% 18% AD-12133 34% 2% 26% 6% AD-12134 18% 3% 14% 2%AD-12135 50% 6% 37% 4% AD-12136 42% 19% 22% 2% AD-12137 85% 12% 92% 4%AD-12138 47% 6% 49% 1% AD-12139 80% 5% 72% 4% AD-12140 97% 22% 67% 9%AD-12141 120% 4% 107% 10% AD-12142 55% 8% 33% 4% AD-12143 64% 34% 19% 2%AD-12144 58% 29% 17% 2% AD-12145 27% 8% 18% 2% AD-12146 19% 20% 15% 1%AD-12147 29% 9% 35% 3% AD-12148 30% 3% 56% 5% AD-12149 8% 2% 12% 3%AD-12150 31% 2% 31% 7% AD-12151 9% 5% 14% 2% AD-12152 3% 3% 23% 3%AD-12153 20% 6% 34% 4% AD-12154 24% 7% 44% 3% AD-12155 33% 6% 53% 11%AD-12156 35% 5% 40% 5% AD-12157 8% 3% 23% 4% AD-12158 13% 2% 22% 5%AD-12159 34% 6% 46% 5% AD-12160 19% 3% 31% 4% AD-12161 88% 4% 83% 7%AD-12162 26% 7% 32% 7% AD-12163 55% 9% 40% 3% AD-12164 21% 3% AD-1216530% 3% 41% 4% AD-12166 9% 10% 22% 9% AD-12167 26% 3% 30% 2% AD-12168 54%4% 59% 20% AD-12169 41% 4% 51% 16% AD-12170 43% 4% 52% 20% AD-12171 67%3% 73% 25% AD-12172 53% 15% 37% 2% AD-12173 39% 0% 39% 0% AD-12174 41%5% 27% 0% AD-12175 29% 0% 38% 14% AD-12176 43% 2% 56% 25% AD-12177 68%6% 74% 30% AD-12178 41% 4% 41% 6% AD-12179 53% 5% 44% 5% AD-12180 16% 2%13% 4% AD-12181 19% 3% 14% 2% AD-12182 16% 4% 18% 8% AD-12183 26% 3% 19%4% AD-12184 54% 2% 77% 8% AD-12185 8% 1% 9% 1% AD-12186 36% 3% 41% 6%AD-12187 34% 17% 27% 1% AD-12188 30% 3% 27% 4% AD-12189 51% 4% 48% 5%AD-12190 33% 2% 26% 4% AD-12191 20% 2% 13% 0% AD-12192 21% 1% 23% 10%AD-12193 64% 8% 98% 6% AD-12194 8% 2% 15% 4% AD-12195 34% 2% 48% 3%AD-12196 34% 2% 51% 3% AD-12197 75% 4% 93% 6% AD-12198 55% 5% 48% 2%AD-12199 102% 6% 118% 9% AD-12200 75% 6% 60% 12% AD-12201 42% 3% 16% 4%AD-12202 29% 4% 9% 3% AD-12203 114% 14% 89% 20% AD-12204 64% 7% 26% 5%AD-12205 66% 12% 35% 4% AD-12206 46% 3% 32% 12% AD-12207 57% 5% 40% 6%AD-12208 30% 8% 10% 5% AD-12209 101% 6% 102% 23% AD-12210 38% 11% 27%14% AD-12211 16% 6% 10% 5% AD-12212 59% 8% 65% 5% AD-12213 24% 9% 12% 2%AD-12214 67% 14% 70% 12% AD-12215 29% 13% 13% 4% AD-12216 36% 4% 13% 1%AD-12217 36% 9% 11% 2% AD-12218 35% 5% 17% 3% AD-12219 41% 9% 14% 1%AD-12220 37% 5% 23% 3% AD-12221 58% 7% 39% 6% AD-12222 74% 9% 53% 3%AD-12223 74% 10% 67% 7% AD-12224 24% 2% 11% 2% AD-12225 75% 5% 76% 14%AD-12226 45% 8% 40% 3% AD-12227 61% 6% 47% 5% AD-12228 28% 3% 25% 5%AD-12229 54% 13% 37% 6% AD-12230 70% 17% 65% 4% AD-12231 32% 12% 22% 6%AD-12232 30% 3% 17% 2% AD-12233 38% 2% 32% 3% AD-12234 90% 5% 95% 7%AD-12235 57% 7% 46% 3% AD-12236 34% 8% 16% 2% AD-12237 42% 9% 32% 8%AD-12238 42% 6% 34% 6% AD-12239 42% 3% 40% 4% AD-12240 47% 6% 36% 5%AD-12241 69% 5% 70% 8% AD-12242 61% 2% 47% 3% AD-12243 26% 7% 15% 1%AD-12244 25% 6% 15% 1% AD-12245 65% 6% 83% 13% AD-12246 29% 7% 31% 6%AD-12247 57% 13% 50% 3% AD-12248 36% 8% 20% 3% 15% 7% AD-12249 44% 3%70% 11% 103% 34% AD-12250 47% 5% 18% 5% 17% 4% AD-12251 121% 28% 35% 8%60% 42% AD-12252 94% 19% 8% 3% 5% 3% AD-12253 94% 33% 42% 8% 49% 27%AD-12254 101% 58% 70% 5% 80% 32% AD-12255 163% 27% 28% 6% 36% 10%AD-12256 112% 62% 18% 3% 9% 4% AD-12257 10% 4% 9% 2% 6% 2% AD-12258 27%9% 18% 3% 20% 6% AD-12259 20% 5% 12% 2% 13% 5% AD-12260 22% 7% 81% 7%65% 13% AD-12261 122% 11% 66% 7% 80% 22% AD-12262 97% 30% 33% 6% 44% 18%AD-12263 177% 57% 85% 11% 84% 15% AD-12264 37% 6% 10% 1% 10% 4% AD-1226540% 8% 17% 1% 20% 10% AD-12266 33% 9% 9% 1% 8% 4% AD-12267 34% 13% 11%1% 6% 2% AD-12268 34% 6% 11% 1% 9% 2% AD-12269 54% 6% 33% 4% 29% 7%AD-12270 52% 5% 29% 4% 27% 6% AD-12271 53% 7% 27% 3% 19% 6% AD-12272 85%15% 57% 7% 51% 16% AD-12273 36% 6% 26% 2% 30% 5% AD-12274 75% 21% 40% 2%50% 19% AD-12275 29% 9% 8% 1% 8% 4% AD-12276 45% 19% 15% 2% 16% 12%AD-12277 58% 17% 32% 2% 55% 14% AD-12278 120% 35% 96% 10% 124% 38%AD-12279 47% 29% 17% 1% 12% 4% AD-12280 2% 0% 3% 1% AD-12281 2% 0% 5% 2%AD-12282 3% 0% 25% 5% AD-12283 3% 1% 35% 4% AD-12284 5% 2% 49% 8%AD-12285 7% 7% 21% 26% AD-12286 28% 34% 12% 7% AD-12287 40% 21% 51% 23%AD-12288 26% 7% 155% 146% AD-12289 43% 21% 220% 131% AD-12290 2% 1% 81%23% AD-12291 4% 1% 70% 3% AD-12292 2% 1% 6% 2% AD-12293 4% 2% 36% 3%AD-12294 10% 6% 38% 3% AD-12295 29% 31% 37% 3% AD-12296 82% 4% 89% 2%AD-12297 75% 3% 65% 2% AD-12298 73% 4% 60% 3% AD-12299 76% 4% 66% 4%AD-12300 36% 4% 15% 1% AD-12301 33% 4% 18% 2% AD-12302 66% 5% 65% 3%AD-12303 35% 6% 17% 2% AD-12304 70% 8% 70% 6% AD-12305 63% 8% 80% 7%AD-12306 23% 6% 20% 3% AD-12307 78% 10% 58% 5% AD-12308 27% 8% 15% 2%AD-12309 58% 11% 42% 3% AD-12310 106% 23% 80% 2% AD-12311 73% 12% 60% 2%AD-12312 39% 3% 36% 3% AD-12313 64% 9% 49% 6% AD-12314 28% 7% 14% 6%AD-12315 31% 7% 13% 2% AD-12316 42% 5% 14% 2% AD-12317 34% 9% 15% 5%AD-12318 46% 4% 28% 4% AD-12319 77% 3% 56% 4% AD-12320 55% 7% 41% 3%AD-12321 21% 3% 10% 2% AD-12322 27% 8% 30% 12% AD-12323 26% 7% 35% 18%AD-12324 27% 8% 27% 14% AD-12325 32% 12% 32% 22% AD-12326 42% 22% 45%41% AD-12327 36% 14% 37% 32% AD-12328 45% 2% 31% 3% AD-12329 61% 4% 34%3% AD-12330 63% 5% 38% 4% AD-12331 50% 2% 26% 5% AD-12332 80% 4% 51% 7%AD-12333 34% 6% 12% 2% AD-12334 27% 2% 18% 3% AD-12335 84% 6% 60% 7%AD-12336 45% 4% 36% 4% AD-12337 30% 7% 19% 2%

TABLE 3 Sequences and analysis of Eg5/KSP dsRNA duplexes single dose SDsscreen @ 2nd SEQ SEQ 25 nM [% screen ID Antisense sequence (5′- IDduplex residual (among Sense sequence (5′-3′) NO. 3′) NO. name mRNA]quadruplicates) ccAuuAcuAcAGuAGcAcuTsT 582 AGUGCuACUGuAGuAAUGGTsT 583AD-14085 19% 1% AucuGGcAAccAuAuuucuTsT 584 AGAAAuAUGGUUGCcAGAUTsT 585AD-14086 38% 1% GAuAGcuAAAuuAAAccAATsT 586 UUGGUUuAAUUuAGCuAUCTsT 587AD-14087 75% 10% AGAuAccAuuAcuAcAGuATsT 588 uACUGuAGuAAUGGuAUCUTsT 589AD-14088 22% 8% GAuuGuucAucAAuuGGcGTsT 590 CGCcAAUUGAUGAAcAAUCTsT 591AD-14089 70% 12% GcuuucuccucGGcucAcuTsT 592 AGuGAGCCGAGGAGAAAGCTsT 593AD-14090 79% 11% GGAGGAuuGGcuGAcAAGATsT 594 UCUUGUcAGCcAAUCCUCCTsT 595AD-14091 29% 3% uAAuGAAGAGuAuAccuGGTsT 596 CcAGGuAuACUCUUcAUuATsT 597AD-14092 23% 2% uuucAccAAAccAuuuGuATsT 598 uAcAAAUGGUUUGGUGAAATsT 599AD-14093 60% 2% cuuAuuAAGGAGuAuAcGGTsT 600 CCGuAuACUCCUuAAuAAGTsT 601AD-14094 11% 3% GAAAucAGAuGGAcGuAAGTsT 602 CUuACGUCcAUCUGAUUUCTsT 603AD-14095 10% 2% cAGAuGucAGcAuAAGcGATsT 604 UCGCUuAUGCUGAcAUCUGTsT 605AD-14096 27% 2% AucuAAcccuAGuuGuAucTsT 606 GAuAcAACuAGGGUuAGAUTsT 607AD-14097 45% 6% AAGAGcuuGuuAAAAucGGTsT 608 CCGAUUUuAAcAAGCUCUUTsT 609AD-14098 50% 10% uuAAGGAGuAuAcGGAGGATsT 610 UCCUCCGuAuACUCCUuAATsT 611AD-14099 12% 4% uuGcAAuGuAAAuAcGuAuTsT 612 AuACGuAUUuAcAUUGcAATsT 613AD-14100 49% 7% ucuAAcccuAGuuGuAuccTsT 614 GGAuAcAACuAGGGUuAGATsT 615AD-14101 36% 1% cAuGuAucuuuuucucGAuTsT 616 AUCGAGAAAAAGAuAcAUGTsT 617AD-14102 49% 3% GAuGucAGcAuAAGcGAuGTsT 618 cAUCGCUuAUGCUGAcAUCTsT 619AD-14103 74% 5% ucccAAcAGGuAcGAcAccTsT 620 GGUGUCGuACCUGUUGGGATsT 621AD-14104 27% 3% uGcucAcGAuGAGuuuAGuTsT 622 ACuAAACUcAUCGUGAGcATsT 623AD-14105 34% 4% AGAGcuuGuuAAAAucGGATsT 624 UCCGAUUUuAAcAAGCUCUTsT 625AD-14106 9% 2% GcGuAcAAGAAcAucuAuATsT 626 uAuAGAUGUUCUUGuACGCTsT 627AD-14107 5% 1% GAGGuuGuAAGccAAuGuuTsT 628 AAcAUUGGCUuAcAACCUCTsT 629AD-14108 15% 1% AAcAGGuAcGAcAccAcAGTsT 630 CUGUGGUGUCGuACCUGUUTsT 631AD-14109 91% 2% AAcccuAGuuGuAucccucTsT 632 GAGGGAuAcAACuAGGGUUTsT 633AD-14110 66% 5% GcAuAAGcGAuGGAuAAuATsT 634 uAUuAUCcAUCGCUuAUGCTsT 635AD-14111 33% 3% AAGcGAuGGAuAAuAccuATsT 636 uAGGuAUuAUCcAUCGCUUTsT 637AD-14112 51% 3% uGAuccuGuAcGAAAAGAATsT 638 UUCUUUUCGuAcAGGAUcATsT 639AD-14113 22% 3% AAAAcAuuGGccGuucuGGTsT 640 CcAGAACGGCcAAUGUUUUTsT 641AD-14114 117% 8% cuuGGAGGGcGuAcAAGAATsT 642 UUCUUGuACGCCCUCcAAGTsT 643AD-14115 50% 8% GGcGuAcAAGAAcAucuAuTsT 644 AuAGAUGUUCUUGuACGCCTsT 645AD-14116 14% 3% AcucuGAGuAcAuuGGAAuTsT 646 AUUCcAAUGuACUcAGAGUTsT 647AD-14117 12% 4% uuAuuAAGGAGuAuAcGGATsT 648 UCCGuAuACUCCUuAAuAATsT 649AD-14118 26% 4% uAAGGAGuAuAcGGAGGAGTsT 650 CUCCUCCGuAuACUCCUuATsT 651AD-14119 24% 5% AAAucAAuAGucAAcuAAATsT 652 UUuAGUUGACuAUUGAUUUTsT 653AD-14120 8% 1% AAucAAuAGucAAcuAAAGTsT 654 CUUuAGUUGACuAUUGAUUTsT 655AD-14121 24% 2% uucucAGuAuAcuGuGuAATsT 656 UuAcAcAGuAuACUGAGAATsT 657AD-14122 10% 1% uGuGAAAcAcucuGAuAAATsT 658 UUuAUcAGAGUGUUUCAcATsT 659AD-14123 8% 1% AGAuGuGAAucucuGAAcATsT 660 UGUUcAGAGAUUcAcAUCUTsT 661AD-14124 9% 2% AGGuuGuAAGccAAuGuuGTsT 662 cAAcAUUGGCUuAcAACCUTsT 663AD-14125 114% 6% uGAGAAAucAGAuGGAcGuTsT 664 ACGUCcAUCUGAUUUCUcATsT 665AD-14126 9% 1% AGAAAucAGAuGGAcGuAATsT 666 UuACGUCcAUCUGAUUUCUTsT 667AD-14127 57% 6% AuAucccAAcAGGuAcGAcTsT 668 GUCGuACCUGUUGGGAuAUTsT 669AD-14128 104% 6% cccAAcAGGuAcGAcAccATsT 670 UGGUGUCGuACCUGUUGGGTsT 671AD-14129 21% 2% AGuAuAcuGAAGAAccucuTsT 672 AGAGGUUCUUcAGuAuACUTsT 673AD-14130 57% 6% AuAuAuAucAGccGGGcGcTsT 674 GCGCCCGGCUGAuAuAuAUTsT 675AD-14131 93% 6% AAucuAAcccuAGuuGuAuTsT 676 AuAcAACuAGGGUuAGAUUTsT 677AD-14132 75% 8% cuAAcccuAGuuGuAucccTsT 678 GGGAuAcAACuAGGGUuAGTsT 679AD-14133 66% 4% cuAGuuGuAucccuccuuuTsT 680 AAAGGAGGGAuAcAACuAGTsT 681AD-14134 44% 6% AGAcAucuGAcuAAuGGcuTsT 682 AGCcAUuAGUcAGAUGUCUTsT 683AD-14135 55% 6% GAAGcucAcAAuGAuuuAATsT 684 UuAAAUcAUUGUGAGCUUCTsT 685AD-14136 29% 3% AcAuGuAucuuuuucucGATsT 686 UCGAGAAAAAGAuAcAUGUTsT 687AD-14137 40% 3% ucGAuucAAAucuuAAcccTsT 688 GGGUuAAGAUUUGAAUCGATsT 689AD-14138 39% 5% ucuuAAcccuuAGGAcucuTsT 690 AGAGUCCuAAGGGUuAAGATsT 691AD-14139 71% 11% GcucAcGAuGAGuuuAGuGTsT 692 cACuAAACUcAUCGUGAGCTsT 693AD-14140 43% 15% cAuAAGcGAuGGAuAAuAcTsT 694 GuAUuAUCcAUCGCUuAUGTsT 695AD-14141 33% 6% AuAAGcGAuGGAuAAuAccTsT 696 GGuAUuAUCcAUCGCUuAUTsT 697AD-14142 51% 14% ccuAAuAAAcuGcccucAGTsT 698 CUGAGGGcAGUUuAUuAGGTsT 699AD-14143 42% 1% ucGGAAAGuuGAAcuuGGuTsT 700 ACcAAGUUcAACUUUCCGATsT 701AD-14144 4% 4% GAAAAcAuuGGccGuucuGTsT 702 cAGAACGGCcAAUGUUUUCTsT 703AD-14145 92% 5% AAGAcuGAucuucuAAGuuTsT 704 AACUuAGAAGAUcAGUCUUTsT 705AD-14146 13% 2% GAGcuuGuuAAAAucGGAATsT 706 UUCCGAUUUuAAcAAGCUCTsT 707AD-14147 8% 1% AcAuuGGccGuucuGGAGcTsT 708 GCUCcAGAACGGCcAAUGUTsT 709AD-14148 80% 7% AAGAAcAucuAuAAuuGcATsT 710 UGcAAUuAuAGAUGUUCUUTsT 711AD-14149 44% 7% AAAuGuGucuAcucAuGuuTsT 712 AAcAUGAGuAGAcAcAUUUTsT 713AD-14150 32% 29% uGucuAcucAuGuuucucATsT 714 UGAGAAAcAUGAGuAGAcATsT 715AD-14151 75% 11% GuAuAcuGuGuAAcAAucuTsT 716 AGAUUGUuAcAcAGuAuACTsT 717AD-14152 8% 5% uAuAcuGuGuAAcAAucuATsT 718 uAGAUUGUuAcAcAGuAuATsT 719AD-14153 17% 11% cuuAGuAGuGuccAGGAAATsT 720 UUUCCUGGAcACuACuAAGTsT 721AD-14154 16% 4% ucAGAuGGAcGuAAGGcAGTsT 722 CUGCCUuACGUCcAUCUGATsT 723AD-14155 11% 1% AGAuAAAuuGAuAGcAcAATsT 724 UUGUGCuAUcAAUUuAUCUTsT 725AD-14156 10% 1% cAAcAGGuAcGAcAccAcATsT 726 UGUGGUGUCGuACCUGUUGTsT 727AD-14157 29% 3% uGcAAuGuAAAuAcGuAuuTsT 728 AAuACGuAUUuAcAUUGcATsT 729AD-14158 51% 3% AGucAGAAuuuuAucuAGATsT 730 UCuAGAuAAAAUUCUGACUTsT 731AD-14159 53% 5% cuAGAAAucuuuuAAcAccTsT 732 GGUGUuAAAAGAUUUCuAGTsT 733AD-14160 40% 3% AAuAAAucuAAcccuAGuuTsT 734 AACuAGGGUuAGAUUuAUUTsT 735AD-14161 83% 7% AAuuuucuGcucAcGAuGATsT 736 UcAUCGUGAGcAGAAAAUUTsT 737AD-14162 44% 6% GcccucAGuAAAuccAuGGTsT 738 CcAUGGAUUuACUGAGGGCTsT 739AD-14163 57% 3% AcGuuuAAAAcGAGAucuuTsT 740 AAGAUCUCGUUUuAAACGUTsT 741AD-14164 4% 1% AGGAGAuAGAAcGuuuAAATsT 742 UUuAAACGUUCuAUCUCCUTsT 743AD-14165 11% 1% GAccGucAuGGcGucGcAGTsT 744 CUGCGACGCcAUGACGGUCTsT 745AD-14166 90% 5% AccGucAuGGcGucGcAGcTsT 746 GCUGCGACGCcAUGACGGUTsT 747AD-14167 49% 1% GAAcGuuuAAAAcGAGAucTsT 748 GAUCUCGUUUuAAACGUUcTsT 749AD-14168 12% 2% uuGAGcuuAAcAuAGGuAATsT 750 UuACCuAUGUuAAGCUcAATsT 751AD-14169 66% 4% AcuAAAuuGAucucGuAGATsT 752 UCuACGAGAUcAAUUuAGUTsT 753AD-14170 52% 6% ucGuAGAAuuAucuuAAuATsT 754 uAUuAAGAuAAUUCuACGATsT 755AD-14171 42% 4% GGAGAuAGAAcGuuuAAAATsT 756 UUUuAAACGUUCuAUCUCCTsT 757AD-14172 3% 1% AcAAcuuAuuGGAGGuuGuTsT 758 AcAACCUCcAAuAAGUUGUTsT 759AD-14173 29% 2% uGAGcuuAAcAuAGGuAAATsT 760 UUuACCuAUGUuAAGCUcATsT 761AD-14174 69% 2% AucucGuAGAAuuAucuuATsT 762 uAAGAuAAUUCuACGAGAUTsT 763AD-14175 53% 3% cuGcGuGcAGucGGuccucTsT 764 GAGGACCGACUGcACGcAGTsT 765AD-14176 111% 4% cAcGcAGcGcccGAGAGuATsT 766 uACUCUCGGGCGCUGCGUGTsT 767AD-14177 87% 6% AGuAccAGGGAGAcuccGGTsT 768 CCGGAGUCUCCCUGGuACUTsT 769AD-14178 59% 2% AcGGAGGAGAuAGAAcGuuTsT 770 AACGUUCuAUCUCCUCCGUTsT 771AD-14179 9% 2% AGAAcGuuuAAAAcGAGAuTsT 772 AUCUCGUUUuAAACGUUCUTsT 773AD-14180 43% 2% AAcGuuuAAAAcGAGAucuTsT 774 AGAUCUCGUUUuAAACGUUTsT 775AD-14181 70% 10% AGcuuGAGcuuAAcAuAGGTsT 776 CCuAUGUuAAGCUcAAGCUTsT 777AD-14182 100% 7% AGcuuAAcAuAGGuAAAuATsT 778 uAUUuACCuAUGUuAAGCUTsT 779AD-14183 60% 5% uAGAGcuAcAAAAccuAucTsT 780 GAuAGGUUUUGuAGCUCuATsT 781AD-14184 129% 6% uAGuuGuAucccuccuuuATsT 782 uAAAGGAGGGAuAcAACuATsT 783AD-14185 62% 4% AccAcccAGAcAucuGAcuTsT 784 AGUcAGAUGUCUGGGUGGUTsT 785AD-14186 42% 3% AGAAAcuAAAuuGAucucGTsT 786 CGAGAUcAAUUuAGUUUCUTsT 787AD-14187 123% 12% ucucGuAGAAuuAucuuAATsT 788 UuAAGAuAAUUCuACGAGATsT 789AD-14188 38% 2% cAAcuuAuuGGAGGuuGuATsT 790 uAcAACCUCcAAuAAGUUGTsT 791AD-14189 13% 1% uuGuAucccuccuuuAAGuTsT 792 ACUuAAAGGAGGGAuAcAATsT 793AD-14190 59% 3% ucAcAAcuuAuuGGAGGuuTsT 794 AACCUCcAAuAAGUUGUGATsT 795AD-14191 93% 3% AGAAcuGuAcucuucucAGTsT 796 CUGAGAAGAGuAcAGUUCUTsT 797AD-14192 45% 5% GAGcuuAAcAuAGGuAAAuTsT 798 AUUuACCuAUGUuAAGCUCTsT 799AD-14193 57% 3% cAccAAcAucuGuccuuAGTsT 800 CuAAGGAcAGAUGUUGGUGTsT 801AD-14194 51% 4% AAAGcccAcuuuAGAGuAuTsT 802 AuACUCuAAAGUGGGCUUUTsT 803AD-14195 77% 5% AAGcccAcuuuAGAGuAuATsT 804 uAuACUCuAAAGUGGGCUUTsT 805AD-14196 42% 6% GAccuuAuuuGGuAAucuGTsT 806 cAGAUuACcAAAuAAGGUCTsT 807AD-14197 15% 2% GAuuAAuGuAcucAAGAcuTsT 808 AGUCUUGAGuAcAUuAAUCTsT 809AD-14198 12% 2% cuuuAAGAGGccuAAcucATsT 810 UGAGUuAGGCCUCUuAAAGTsT 811AD-14199 18% 2% uuAAAccAAAcccuAuuGATsT 812 UcAAuAGGGUUUGGUUuAATsT 813AD-14200 72% 9% ucuGuuGGAGAucuAuAAuTsT 814 AUuAuAGAUCUCcAAcAGATsT 815AD-14201 9% 3% cuGAuGuuucuGAGAGAcuTsT 816 AGUCUCUcAGAAAcAUcAGTsT 817AD-14202 25% 3% GcAuAcucuAGucGuucccTsT 818 GGGAACGACuAGAGuAUGCTsT 819AD-14203 21% 1% GuuccuuAucGAGAAucuATsT 820 uAGAUUCUCGAuAAGGAACTsT 821AD-14204 4% 2% GcAcuuGGAucucucAcAuTsT 822 AUGUGAGAGAUCcAAGUGCTsT 823AD-14205 5% 1% AAAAAAGGAAcuAGAuGGcTsT 824 GCcAUCuAGUUCCUUUUUUTsT 825AD-14206 79% 6% AGAGcAGAuuAccucuGcGTsT 826 CGcAGAGGuAAUCUGCUCUTsT 827AD-14207 55% 2% AGcAGAuuAccucuGcGAGTsT 828 CUCGcAGAGGuAAUCUGCUTsT 829AD-14208 100% 4% cccuGAcAGAGuucAcAAATsT 830 UUUGUGAACUCUGUcAGGGTsT 831AD-14209 34% 3% GuuuAccGAAGuGuuGuuuTsT 832 AAAcAAcACUUCGGuAAACTsT 833AD-14210 13% 2% uuAcAGuAcAcAAcAAGGATsT 834 UCCUUGUUGUGuACUGuAATsT 835AD-14211 9% 1% AcuGGAucGuAAGAAGGcATsT 836 UGCCUUCUuACGAUCcAGUTsT 837AD-14212 20% 3% GAGcAGAuuAccucuGcGATsT 838 UCGcAGAGGuAAUCUGCUCTsT 839AD-14213 48% 5% AAAAGAAGuuAGuGuAcGATsT 840 UCGuAcACuAACUUCUUUUTsT 841AD-14214 28% 18% GAccAuuuAAuuuGGcAGATsT 842 UCUGCcAAAUuAAAUGGUCTsT 843AD-14215 132% 0% GAGAGGAGuGAuAAuuAAATsT 844 UUuAAUuAUcACUCCUCUCTsT 845AD-14216 3% 0% cuGGAGGAuuGGcuGAcAATsT 846 UUGUcAGCcAAUCCUCcAGTsT 847AD-14217 19% 1% cucuAGucGuucccAcucATsT 848 UGAGUGGGAACGACuAGAGTsT 849AD-14218 67% 8% GAuAccAuuAcuAcAGuAGTsT 850 CuACUGuAGuAAUGGuAUCTsT 851AD-14219 76% 4% uucGucuGcGAAGAAGAAATsT 852 UUUCUUCUUCGcAGACGAATsT 853AD-14220 33% 8% GAAAAGAAGuuAGuGuAcGTsT 854 CGuAcACuAACUUCUUUUCTsT 855AD-14221 25% 2% uGAuGuuuAccGAAGuGuuTsT 856 AAcACUUCGGuAAAcAUcATsT 857AD-14222 7% 2% uGuuuGuccAAuucuGGAuTsT 858 AUCcAGAAUUGGAcAAAcATsT 859AD-14223 19% 2% AuGAAGAGuAuAccuGGGATsT 860 UCCcAGGuAuACUCUUcAUTsT 861AD-14224 13% 1% GcuAcucuGAuGAAuGcAuTsT 862 AUGcAUUcAUcAGAGuAGCTsT 863AD-14225 15% 2% GcccuuGuAGAAAGAAcAcTsT 864 GUGUUCUUUCuAcAAGGGCTsT 865AD-14226 11% 0% ucAuGuuccuuAucGAGAATsT 866 UUCUCGAuAAGGAAcAUGATsT 867AD-14227 5% 1% GAAuAGGGuuAcAGAGuuGTsT 868 cAACUCUGuAACCCuAUUCTsT 869AD-14228 34% 3% cAAAcuGGAucGuAAGAAGTsT 870 CUUCUuACGAUCcAGUUUGTsT 871AD-14229 15% 2% cuuAuuuGGuAAucuGcuGTsT 872 cAGcAGAUuACcAAAuAAGTsT 873AD-14230 20% 1% AGcAAuGuGGAAAccuAAcTsT 874 GUuAGGUUUCcAcAUUGCUTsT 875AD-14231 18% 1% AcAAuAAAGcAGAcccAuuTsT 876 AAUGGGUCUGCUUuAUUGUTsT 877AD-14232 21% 1% AAccAcuuAGuAGuGuccATsT 878 UGGAcACuACuAAGUGGUUTsT 879AD-14233 106% 12% AGucAAGAGccAucuGuAGTsT 880 CuAcAGAUGGCUCUUGACUTsT 881AD-14234 35% 3% cucccuAGAcuucccuAuuTsT 882 AAuAGGGAAGUCuAGGGAGTsT 883AD-14235 48% 4% AuAGcuAAAuuAAAccAAATsT 884 UUUGGUUuAAUUuAGCuAUTsT 885AD-14236 23% 3% uGGcuGGuAuAAuuccAcGTsT 886 CGUGGAAUuAuACcAGCcATsT 887AD-14237 79% 9% uuAuuuGGuAAucuGcuGuTsT 888 AcAGcAGAUuACcAAAuAATsT 889AD-14238 92% 7% AAcuAGAuGGcuuucucAGTsT 890 CUGAGAAAGCcAUCuAGUUTsT 891AD-14239 20% 2% ucAuGGcGucGcAGccAAATsT 892 UUUGGCUGCGACGCcAUGATsT 893AD-14240 71% 6% AcuGGAGGAuuGGcuGAcATsT 894 UGUcAGCcAAUCCUCcAGUTsT 895AD-14241 14% 1% cuAuAAuuGcAcuAucuuuTsT 896 AAAGAuAGUGcAAUuAuAGTsT 897AD-14242 11% 2% AAAGGucAccuAAuGAAGATsT 898 UCUUcAUuAGGUGACCUUUTsT 899AD-14243 11% 1% AuGAAuGcAuAcucuAGucTsT 900 GACuAGAGuAUGcAUUcAUTsT 901AD-14244 15% 2% AAcAuAuuGAAuAAGccuGTsT 902 cAGGCUuAUUcAAuAUGUUTsT 903AD-14245 80% 7% AAGAAGGcAGuuGAccAAcTsT 904 GUUGGUcAACUGCCUUCUUTsT 905AD-14246 57% 5% GAuAcuAAAAGAAcAAucATsT 906 UGAUUGUUCUUUuAGuAUCTsT 907AD-14247 9% 3% AuAcuGAAAAucAAuAGucTsT 908 GACuAUUGAUUUUcAGuAUTsT 909AD-14248 39% 4% AAAAAGGAAcuAGAuGGcuTsT 910 AGCcAUCuAGUUCCUUUUUTsT 911AD-14249 64% 2% GAAcuAGAuGGcuuucucATsT 912 UGAGAAAGCcAUCuAGUUCTsT 913AD-14250 18% 2% GAAAccuAAcuGAAGAccuTsT 914 AGGUCUUcAGUuAGGUUUCTsT 915AD-14251 56% 6% uAcccAucAAcAcuGGuAATsT 916 UuACcAGUGUUGAUGGGuATsT 917AD-14252 48% 6% AuuuuGAuAucuAcccAuuTsT 918 AAUGGGuAGAuAUcAAAAUTsT 919AD-14253 39% 5% AucccuAuAGuucAcuuuGTsT 920 cAAAGUGAACuAuAGGGAUTsT 921AD-14254 44% 8% AuGGGcuAuAAuuGcAcuATsT 922 uAGUGcAAUuAuAGCCcAUTsT 923AD-14255 108% 8% AGAuuAccucuGcGAGcccTsT 924 GGGCUCGcAGAGGuAAUCUTsT 925AD-14256 108% 6% uAAuuccAcGuAcccuucATsT 926 UGAAGGGuACGUGGAAUuATsT 927AD-14257 23% 2% GucGuucccAcucAGuuuuTsT 928 AAAACuGAGuGGGAACGACTsT 929AD-14258 21% 3% AAAucAAucccuGuuGAcuTsT 930 AGUcAAcAGGGAUUGAUUUTsT 931AD-14259 19% 2% ucAuAGAGcAAAGAAcAuATsT 932 uAUGUUCUUUGCUCuAUGATsT 933AD-14260 10% 1% uuAcuAcAGuAGcAcuuGGTsT 934 CcAAGUGCuACUGuAGuAATsT 935AD-14261 76% 3% AuGuGGAAAccuAAcuGAATsT 936 UUcAGUuAGGUUUCcAcAUTsT 937AD-14262 13% 2% uGuGGAAAccuAAcuGAAGTsT 938 CUUcAGUuAGGUUUCcAcATsT 939AD-14263 14% 2% ucuuccuuAAAuGAAAGGGTsT 940 CCCUUUcAUUuAAGGAAGATsT 941AD-14264 65% 3% uGAAGAAccucuAAGucAATsT 942 UUGACUuAGAGGUUCUUcATsT 943AD-14265 13% 1% AGAGGucuAAAGuGGAAGATsT 944 UCUUCcACUUuAGACCUCUTsT 945AD-14266 18% 3% AuAucuAcccAuuuuucuGTsT 946 cAGAAAAAUGGGuAGAuAUTsT 947AD-14267 50% 9% uAAGccuGAAGuGAAucAGTsT 948 CUGAUUcACUUcAGGCUuATsT 949AD-14268 13% 3% AGAuGcAGAccAuuuAAuuTsT 950 AAUuAAAUGGUCUGcAUCUTsT 951AD-14269 19% 4% AGuGuuGuuuGuccAAuucTsT 952 GAAUUGGAcAAAcAAcACUTsT 953AD-14270 11% 2% cuAuAAuGAAGAGcuuuuuTsT 954 AAAAAGCUCUUcAUuAuAGTsT 955AD-14271 11% 1% AGAGGAGuGAuAAuuAAAGTsT 956 CUUuAAUuAUcACUCCUCUTsT 957AD-14272 7% 1% uuucucuGuuAcAAuAcAuTsT 958 AUGuAUUGuAAcAGAGAAATsT 959AD-14273 14% 2% AAcAucuAuAAuuGcAAcATsT 960 UGUUGcAAUuAuAGAUGUUTsT 961AD-14274 73% 4% uGcuAGAAGuAcAuAAGAcTsT 962 GUCUuAUGuACUUCuAGcATsT 963AD-14275 10% 1% AAuGuAcucAAGAcuGAucTsT 964 GAUcAGUCUUGAGuAcAUUTsT 965AD-14276 89% 2% GuAcucAAGAcuGAucuucTsT 966 GAAGAUcAGUCUUGAGuACTsT 967AD-14277 7% 1% cAcucuGAuAAAcucAAuGTsT 968 cAUUGAGUUuAUcAGAGUGTsT 969AD-14278 12% 1% AAGAGcAGAuuAccucuGcTsT 970 GcAGAGGuAAUCUGCUCUUTsT 971AD-14279 104% 3% ucuGcGAGcccAGAucAAcTsT 972 GUUGAUCUGGGCUCGcAGATsT 973AD-14280 21% 2% AAcuuGAGccuuGuGuAuATsT 974 uAuAcAcAAGGCUcAAGUUTsT 975AD-14281 43% 3% GAAuAuAuAuAucAGccGGTsT 976 CCGGCUGAuAuAuAuAUUCTsT 977AD-14282 45% 6% uGucAucccuAuAGuucAcTsT 978 GUGAACuAuAGGGAUGAcATsT 979AD-14283 35% 5% GAucuGGcAAccAuAuuucTsT 980 GAAAuAUGGUUGCcAGAUCTsT 981AD-14284 58% 3% uGGcAAccAuAuuucuGGATsT 982 UCcAGAAAuAUGGUUGCcATsT 983AD-14285 48% 3% GAuGuuuAccGAAGuGuuGTsT 984 cAAcACUUCGGuAAAcAUCTsT 985AD-14286 49% 3% uuccuuAucGAGAAucuAATsT 986 UuAGAUUCUCGAuAAGGAATsT 987AD-14287 6% 1% AGcuuAAuuGcuuucuGGATsT 988 UCcAGAAAGcAAUuAAGCUTsT 989AD-14288 50% 2% uuGcuAuuAuGGGAGAccATsT 990 UGGUCUCCcAuAAuAGcAATsT 991AD-14289 48% 1% GucAuGGcGucGcAGccAATsT 992 UUGGCUGCGACGCcAUGACTsT 993AD-14290 112% 7% uAAuuGcAcuAucuuuGcGTsT 994 CGcAAAGAuAGUGcAAUuATsT 995AD-14291 77% 2% cuAucuuuGcGuAuGGccATsT 996 UGGCcAuACGcAAAGAuAGTsT 997AD-14292 80% 6% ucccuAuAGuucAcuuuGuTsT 998 AcAAAGUGAACuAuAGGGATsT 999AD-14293 58% 2% ucAAccuuuAAuucAcuuGTsT 1000 cAAGUGAAUuAAAGGUUGATsT 1001AD-14294 77% 2% GGcAAccAuAuuucuGGAATsT 1002 UUCcAGAAAuAUGGUUGCCTsT 1003AD-14295 62% 2% AuGuAcucAAGAcuGAucuTsT 1004 AGAUcAGUCUUGAGuAcAUTsT 1005AD-14296 59% 4% GcAGAccAuuuAAuuuGGcTsT 1006 GCcAAAUuAAAUGGUCUGCTsT 1007AD-14297 37% 1% ucuGAGAGAcuAcAGAuGuTsT 1008 AcAUCUGuAGUCUCUcAGATsT 1009AD-14298 21% 1% uGcucAuAGAGcAAAGAAcTsT 1010 GUUCUUUGCUCuAUGAGcATsT 1011AD-14299 6% 1% AcAuAAGAccuuAuuuGGuTsT 1012 ACcAAAuAAGGUCUuAUGUTsT 1013AD-14300 17% 2% uuuGuGcuGAuucuGAuGGTsT 1014 CcAUcAGAAUcAGcAcAAATsT 1015AD-14301 97% 6% ccAucAAcAcuGGuAAGAATsT 1016 UUCUuACcAGUGUUGAUGGTsT 1017AD-14302 13% 1% AGAcAAuuccGGAuGuGGATsT 1018 UCcAcAUCCGGAAUUGUCUTsT 1019AD-14303 13% 3% GAAcuuGAGccuuGuGuAuTsT 1020 AuAcAcAAGGCUcAAGUUCTsT 1021AD-14304 38% 2% uAAuuuGGcAGAGcGGAAATsT 1022 UUUCCGCUCUGCcAAAUuATsT 1023AD-14305 14% 2% uGGAuGAAGuuAuuAuGGGTsT 1024 CCcAuAAuAACUUcAUCcATsT 1025AD-14306 22% 4% AucuAcAuGAAcuAcAAGATsT 1026 UCUUGuAGUUcAUGuAGAUTsT 1027AD-14307 26% 6% GGuAuuuuuGAucuGGcAATsT 1028 UUGCcAGAUcAAAAAuACCTsT 1029AD-14308 62% 8% cuAAuGAAGAGuAuAccuGTsT 1030 cAGGuAuACUCUUcAUuAGTsT 1031AD-14309 52% 5% uuuGAGAAAcuuAcuGAuATsT 1032 uAUcAGuAAGUUUCUcAAATsT 1033AD-14310 32% 3% cGAuAAGAuAGAAGAucAATsT 1034 UUGAUCUUCuAUCUuAUCGTsT 1035AD-14311 23% 2% cuGGcAAccAuAuuucuGGTsT 1036 CcAGAAAuAUGGUUGCcAGTsT 1037AD-14312 49% 6% uAGAuAccAuuAcuAcAGuTsT 1038 ACUGuAGuAAUGGuAUCuATsT 1039AD-14313 69% 4% GuAuuAAAuuGGGuuucAuTsT 1040 AUGAAACCcAAUUuAAuACTsT 1041AD-14314 52% 3% AAGAccuuAuuuGGuAAucTsT 1042 GAUuACcAAAuAAGGUCUUTsT 1043AD-14315 66% 4% GcuGuuGAuAAGAGAGcucTsT 1044 GAGCUCUCUuAUcAAcAGCTsT 1045AD-14316 19% 4% uAcucAuGuuucucAGAuuTsT 1046 AAUCUGAGAAAcAUGAGuATsT 1047AD-14317 16% 5% cAGAuGGAcGuAAGGcAGcTsT 1048 GCUGCCUuACGUCcAUCUGTsT 1049AD-14318 52% 11% uAucccAAcAGGuAcGAcATsT 1050 UGUCGuACCUGUUGGGAuATsT 1051AD-14319 28% 11% cAuuGcuAuuAuGGGAGAcTsT 1052 GUCUCCcAuAAuAGcAAUGTsT 1053AD-14320 52% 10% cccucAGuAAAuccAuGGuTsT 1054 ACcAUGGAUUuACUGAGGGTsT 1055AD-14321 53% 6% GGucAuuAcuGcccuuGuATsT 1056 uAcAAGGGcAGuAAUGACCTsT 1057AD-14322 20% 2% AAccAcucAAAAAcAuuuGTsT 1058 cAAAUGUUUUUGAGUGGUUTsT 1059AD-14323 116% 6% uuuGcAAGuuAAuGAAucuTsT 1060 AGAUUcAUuAACUUGcAAATsT 1061AD-14324 14% 2% uuAuuuucAGuAGucAGAATsT 1062 UUCUGACuACUGAAAAuAATsT 1063AD-14325 50% 2% uuuucucGAuucAAAucuuTsT 1064 AAGAUUuGAAUCGAGAAAATsT 1065AD-14326 47% 3% GuAcGAAAAGAAGuuAGuGTsT 1066 cACuAACUUCUUUUCGuACTsT 1067AD-14327 18% 2% uuuAAAAcGAGAucuuGcuTsT 1068 AGcAAGAUCUCGUUUuAAATsT 1069AD-14328 19% 1% GAAuuGAuuAAuGuAcucATsT 1070 UGAGuAcAUuAAUcAAUUCTsT 1071AD-14329 94% 10% GAuGGAcGuAAGGcAGcucTsT 1072 GAGCUGCCUuACGUCcAUCTsT 1073AD-14330 60% 4% cAucuGAcuAAuGGcucuGTsT 1074 cAGAGCcAUuAGUcAGAUGTsT 1075AD-14331 54% 7% GuGAuccuGuAcGAAAAGATsT 1076 UCUUUUCGuAcAGGAUcACTsT 1077AD-14332 22% 4% AGcucuuAuuAAGGAGuAuTsT 1078 AuACUCCUuAAuAAGAGCUTsT 1079AD-14333 70% 10% GcucuuAuuAAGGAGuAuATsT 1080 uAuACUCCUuAAuAAGAGCTsT 1081AD-14334 18% 3% ucuuAuuAAGGAGuAuAcGTsT 1082 CGuAuACUCCUuAAuAAGATsT 1083AD-14335 38% 6% uAuuAAGGAGuAuAcGGAGTsT 1084 CUCCGuAuACUCCUuAAuATsT 1085AD-14336 16% 3% cuGcAGcccGuGAGAAAAATsT 1086 UUUUUCUcACGGGCUGcAGTsT 1087AD-14337 65% 4% ucAAGAcuGAucuucuAAGTsT 1088 CUuAGAAGAUcAGUCUUGATsT 1089AD-14338 18% 0% cuucuAAGuucAcuGGAAATsT 1090 UUUCcAGUGAACUuAGAAGTsT 1091AD-14339 20% 4% uGcAAGuuAAuGAAucuuuTsT 1092 AAAGAUUcAUuAACUUGcATsT 1093AD-14340 24% 1% AAucuAAGGAuAuAGucAATsT 1094 UUGACuAuAUCCUuAGAUUTsT 1095AD-14341 27% 3% AucucuGAAcAcAAGAAcATsT 1096 UGUUCUUGUGUUcAGAGAUTsT 1097AD-14342 13% 1% uucuGAAcAGuGGGuAucuTsT 1098 AGAuACCcACUGUUcAGAATsT 1099AD-14343 19% 1% AGuuAuuuAuAcccAucAATsT 1100 UUGAUGGGuAuAAAuAACUTsT 1101AD-14344 23% 2% AuGcuAAAcuGuucAGAAATsT 1102 UUUCUGAAcAGUUuAGcAUTsT 1103AD-14345 21% 4% cuAcAGAGcAcuuGGuuAcTsT 1104 GuAACcAAGUGCUCUGuAGTsT 1105AD-14346 18% 2% uAuAuAucAGccGGGcGcGTsT 1106 CGCGCCCGGCUGAuAuAuATsT 1107AD-14347 67% 2% AuGuAAAuAcGuAuuucuATsT 1108 uAGAAAuACGuAUUuAcAUTsT 1109AD-14348 39% 3% uuuuucucGAuucAAAucuTsT 1110 AGAUUuGAAUCGAGAAAAATsT 1111AD-14349 83% 6% AAucuuAAcccuuAGGAcuTsT 1112 AGUCCuAAGGGUuAAGAUUTsT 1113AD-14350 54% 2% ccuuAGGAcucuGGuAuuuTsT 1114 AAAuACcAGAGUCCuAAGGTsT 1115AD-14351 57% 8% AAuAAAcuGcccucAGuAATsT 1116 UuACUGAGGGcAGUUuAUUTsT 1117AD-14352 82% 3% GAuccuGuAcGAAAAGAAGTsT 1118 CUUCUUUUCGuAcAGGAUCTsT 1119AD-14353 2% 1% AAuGuGAuccuGuAcGAAATsT 1120 UUUCGuAcAGGAUcAcAUUTsT 1121AD-14354 18% 11% GuGAAAAcAuuGGccGuucTsT 1122 GAACGGCcAAUGUUUUcACTsT 1123AD-14355 2% 1% cuuGAGGAAAcucuGAGuATsT 1124 uACUcAGAGUUUCCUcAAGTsT 1125AD-14356 8% 2% cGuuuAAAAcGAGAucuuGTsT 1126 cAAGAUCUCGUUUuAAACGTsT 1127AD-14357 6% 3% uuAAAAcGAGAucuuGcuGTsT 1128 cAGcAAGAUCUCGUUUuAATsT 1129AD-14358 98% 17% AAAGAuGuAucuGGucuccTsT 1130 GGAGACcAGAuAcAUCUUUTsT 1131AD-14359 10% 1% cAGAAAAuGuGucuAcucATsT 1132 UGAGuAGAcAcAUUUUCUGTsT 1133AD-14360 6% 4% cAGGAAuuGAuuAAuGuAcTsT 1134 GuAcAUuAAUcAAUUCCUGTsT 1135AD-14361 30% 5% AGucAAcuAAAGcAuAuuuTsT 1136 AAAuAUGCUUuAGUUGACUTsT 1137AD-14362 28% 2% uGuGuAAcAAucuAcAuGATsT 1138 UcAUGuAGAUUGUuAcAcATsT 1139AD-14363 60% 6% AuAccAuuuGuuccuuGGuTsT 1140 ACcAAGGAAcAAAUGGuAUTsT 1141AD-14364 12% 9% GcAGAAAucuAAGGAuAuATsT 1142 uAuAUCCUuAGAUUUCUGCTsT 1143AD-14365 5% 2% uGGcuucucAcAGGAAcucTsT 1144 GAGUUCCUGUGAGAAGCcATsT 1145AD-14366 28% 5% GAGAuGuGAAucucuGAAcTsT 1146 GUUcAGAGAUUcAcAUCUCTsT 1147AD-14367 42% 4% uGuAAGccAAuGuuGuGAGTsT 1148 CUcAcAAcAUUGGCUuAcATsT 1149AD-14368 93% 12% AGccAAuGuuGuGAGGcuuTsT 1150 AAGCCUcAcAAcAUUGGCUTsT 1151AD-14369 65% 4% uuGuGAGGcuucAAGuucATsT 1152 UGAACUUGAAGCCUcAcAATsT 1153AD-14370 5% 2% AGGcAGcucAuGAGAAAcATsT 1154 UGUUUCUcAUGAGCUGCCUTsT 1155AD-14371 54% 5% AuAAAuuGAuAGcAcAAAATsT 1156 UUUUGUGCuAUcAAUUuAUTsT 1157AD-14372 4% 1% AcAAAAucuAGAAcuuAAuTsT 1158 AUuAAGUUCuAGAUUUUGUTsT 1159AD-14373 5% 1% GAuAucccAAcAGGuAcGATsT 1160 UCGuACCUGUUGGGAuAUCTsT 1161AD-14374 92% 6% AAGuuAuuuAuAcccAucATsT 1162 UGAUGGGuAuAAAuAACUUTsT 1163AD-14375 76% 4% uGuAAAuAcGuAuuucuAGTsT 1164 CuAGAAAuACGuAUUuAcATsT 1165AD-14376 70% 5% ucuAGuuuucAuAuAAAGuTsT 1166 ACUUuAuAUGAAAACuAGATsT 1167AD-14377 48% 4% AuAAAGuAGuucuuuuAuATsT 1168 uAuAAAAGAACuACUUuAUTsT 1169AD-14378 48% 3% ccAuuuGuAGAGcuAcAAATsT 1170 UUUGuAGCUCuAcAAAUGGTsT 1171AD-14379 44% 5% uAuuuucAGuAGucAGAAuTsT 1172 AUUCUGACuACUGAAAAuATsT 1173AD-14380 35% 16% AAAucuAAcccuAGuuGuATsT 1174 uAcAACuAGGGUuAGAUUUTsT 1175AD-14381 44% 5% cuuuAGAGuAuAcAuuGcuTsT 1176 AGcAAUGuAuACUCuAAAGTsT 1177AD-14382 28% 1% AucuGAcuAAuGGcucuGuTsT 1178 AcAGAGCcAUuAGUcAGAUTsT 1179AD-14383 55% 11% cAcAAuGAuuuAAGGAcuGTsT 1180 cAGUCCUuAAAUcAUUGUGTsT 1181AD-14384 48% 9% ucuuuuucucGAuucAAAuTsT 1182 AUUuGAAUCGAGAAAAAGATsT 1183AD-14385 36% 2% cuuuuucucGAuucAAAucTsT 1184 GAUUuGAAUCGAGAAAAAGTsT 1185AD-14386 41% 7% AuuuucuGcucAcGAuGAGTsT 1186 CUcAUCGUGAGcAGAAAAUTsT 1187AD-14387 38% 3% uuucuGcucAcGAuGAGuuTsT 1188 AACUcAUCGUGAGcAGAAATsT 1189AD-14388 50% 4% AGAGcuAcAAAAccuAuccTsT 1190 GGAuAGGUUUUGuAGCUCUTsT 1191AD-14389 98% 6% GAGccAAAGGuAcAccAcuTsT 1192 AGUGGUGuACCUUUGGCUCTsT 1193AD-14390 43% 8% GccAAAGGuAcAccAcuAcTsT 1194 GuAGUGGUGuACCUUUGGCTsT 1195AD-14391 48% 4% GAAcuGuAcucuucucAGcTsT 1196 GCUGAGAAGAGuAcAGUUCTsT 1197AD-14392 44% 3% AGGuAAAuAucAccAAcAuTsT 1198 AUGUUGGUGAuAUUuACCUTsT 1199AD-14393 37% 2% AGcuAcAAAAccuAuccuuTsT 1200 AAGGAuAGGUUUUGuAGCUTsT 1201AD-14394 114% 7% uGuGAAAGcAuuuAAuuccTsT 1202 GGAAUuAAAUGCUUUcAcATsT 1203AD-14395 55% 4% GcccAcuuuAGAGuAuAcATsT 1204 UGuAuACUCuAAAGUGGGCTsT 1205AD-14396 49% 5% uGuGccAcAcuccAAGAccTsT 1206 GGUCUUGGAGUGUGGcAcATsT 1207AD-14397 71% 6% AAAcuAAAuuGAucucGuATsT 1208 uACGAGAUcAAUUuAGUUUTsT 1209AD-14398 81% 7% uGAucucGuAGAAuuAucuTsT 1210 AGAuAAUUCuACGAGAUcATsT 1211AD-14399 38% 4% GcGuGcAGucGGuccuccATsT 1212 UGGAGGACCGACUGcACGCTsT 1213AD-14400 106% 8% AAAGuuuAGAGAcAucuGATsT 1214 UcAGAUGUCUCuAAACUUUTsT 1215AD-14401 47% 3% cAGAAGGAAuAuGuAcAAATsT 1216 UUUGuAcAuAUUCCUUCUGTsT 1217AD-14402 31% 1% cGcccGAGAGuAccAGGGATsT 1218 UCCCUGGuACUCUCGGGCGTsT 1219AD-14403 105% 4% cGGAGGAGAuAGAAcGuuuTsT 1220 AAACGUUCuAUCUCCUCCGTsT 1221AD-14404 3% 1% AGAuAGAAcGuuuAAAAcGTsT 1222 CGUUUuAAACGUUCuAUCUTsT 1223AD-14405 15% 1% GGAAcAGGAAcuucAcAAcTsT 1224 GUuGuGAAGUUCCuGUUCCTsT 1225AD-14406 44% 5% GuGAGccAAAGGuAcAccATsT 1226 UGGUGuACCUUUGGCUcACTsT 1227AD-14407 41% 4% AuccucccuAGAcuucccuTsT 1228 AGGGAAGUCuAGGGAGGAUTsT 1229AD-14408 104% 3% cAcAcuccAAGAccuGuGcTsT 1230 GcAcAGGUCUUGGAGUGUGTsT 1231AD-14409 67% 4% AcAGAAGGAAuAuGuAcAATsT 1232 UUGuAcAuAUUCCUUCUGUTsT 1233AD-14410 22% 1% uuAGAGAcAucuGAcuuuGTsT 1234 cAAAGUcAGAUGUCUCuAATsT 1235AD-14411 29% 3% AAuuGAucucGuAGAAuuATsT 1236 uAAUUCuACGAGAUcAAUUTsT 1237AD-14412 31% 4%

dsRNA Targeting the VEGF Gene

Four hundred target sequences were identified within exons 1-5 of theVEGF-A121 mRNA sequence. reference transcript is: NM_(—)003376.

(SEQ ID NO: 1539) 1 augaacuuuc ugcugucuug ggugcauugg agccuugccuugcugcucua ccuccaccau 61 gccaaguggu cccaggcugc acccauggca gaaggaggagggcagaauca ucacgaagug 121 gugaaguuca uggaugucua ucagcgcagc uacugccauccaaucgagac ccugguggac 181 aucuuccagg aguacccuga ugagaucgag uacaucuucaagccauccug ugugccccug 241 augcgaugcg ggggcugcug caaugacgag ggccuggagugugugcccac ugaggagucc 301 aacaucacca ugcagauuau gcggaucaaa ccucaccaaggccagcacau aggagagaug 361 agcuuccuac agcacaacaa augugaaugc agaccaaagaaagauagagc aagacaagaa 421 aaaugugaca agccgaggcg guga

Table 4a includes the identified target sequences. Corresponding siRNAstargeting these sequences were subjected to a bioinformatics screen.

To ensure that the sequences were specific to VEGF sequence and not tosequences from any other genes, the target sequences were checkedagainst the sequences in Genbank using the BLAST search engine providedby NCBI. The use of the BLAST algorithm is described in Altschul et al.,J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol.266:460, 1996.

siRNAs were also prioritized for their ability to cross react withmonkey, rat and human VEGF sequences.

Of these 400 potential target sequences 80 were selected for analysis byexperimental screening in order to identify a small number of leadcandidates. A total of 114 siRNA molecules were designed for these 80target sequences 114 (Table 4b).

TABLE 4a Target sequences in VEGF-121 position TARGET SEQUENCE IN SEQ IDin VEGF- VEGF121 mRNA NO: 121 ORF 5′ to 3′ 1540 1AUGAACUUUCUGCUGUCUUGGGU 1541 2 UGAACUUUCUGCUGUCUUGGGUG 1542 3GAACUUUCUGCUGUCUUGGGUGC 1543 4 AACUUUCUGCUGUCUUGGGUGCA 1544 5ACUUUCUGCUGUCUUGGGUGCAU 1545 6 CUUUCUGCUGUCUUGGGUGCAUU 1546 7UUUCUGCUGUCUUGGGUGCAUUG 1547 8 UUCUGCUGUCUUGGGUGCAUUGG 1548 9UCUGCUGUCUUGGGUGCAUUGGA 1549 10 CUGCUGUCUUGGGUGCAUUGGAG 1550 11UGCUGUCUUGGGUGCAUUGGAGC 1551 12 GCUGUCUUGGGUGCAUUGGAGCC 1552 13CUGUCUUGGGUGCAUUGGAGCCU 1553 14 UGUCUUGGGUGCAUUGGAGCCUU 1554 15GUCUUGGGUGCAUUGGAGCCUUG 1555 16 UCUUGGGUGCAUUGGAGCCUUGC 1556 17CUUGGGUGCAUUGGAGCCUUGCC 1557 18 UUGGGUGCAUUGGAGCCUUGCCU 1558 19UGGGUGCAUUGGAGCCUUGCCUU 1559 20 GGGUGCAUUGGAGCCUUGCCUUG 1560 21GGUGCAUUGGAGCCUUGCCUUGC 1561 22 GUGCAUUGGAGCCUUGCCUUGCU 1562 23UGCAUUGGAGCCUUGCCUUGCUG 1563 24 GCAUUGGAGCCUUGCCUUGCUGC 1564 25CAUUGGAGCCUUGCCUUGCUGCU 1565 26 AUUGGAGCCUUGCCUUGCUGCUC 1566 27UUGGAGCCUUGCCUUGCUGCUCU 1567 28 UGGAGCCUUGCCUUGCUGCUCUA 1568 29GGAGCCUUGCCUUGCUGCUCUAC 1569 30 GAGCCUUGCCUUGCUGCUCUACC 1570 31AGCCUUGCCUUGCUGCUCUACCU 1571 32 GCCUUGCCUUGCUGCUCUACCUC 1572 33CCUUGCCUUGCUGCUCUACCUCC 1573 34 CUUGCCUUGCUGCUCUACCUCCA 1574 35UUGCCUUGCUGCUCUACCUCCAC 1575 36 UGCCUUGCUGCUCUACCUCCACC 1576 37GCCUUGCUGCUCUACCUCCACCA 1577 38 CCUUGCUGCUCUACCUCCACCAU 1578 39CUUGCUGCUCUACCUCCACCAUG 1579 40 UUGCUGCUCUACCUCCACCAUGC 1580 41UGCUGCUCUACCUCCACCAUGCC 1581 42 GCUGCUCUACCUCCACCAUGCCA 1582 43CUGCUCUACCUCCACCAUGCCAA 1583 44 UGCUCUACCUCCACCAUGCCAAG 1584 45GCUCUACCUCCACCAUGCCAAGU 1585 46 CUCUACCUCCACCAUGCCAAGUG 1586 47UCUACCUCCACCAUGCCAAGUGG 1587 48 CUACCUCCACCAUGCCAAGUGGU 1588 49UACCUCCACCAUGCCAAGUGGUC 1589 50 ACCUCCACCAUGCCAAGUGGUCC 1590 51CCUCCACCAUGCCAAGUGGUCCC 1591 52 CUCCACCAUGCCAAGUGGUCCCA 1592 53UCCACCAUGCCAAGUGGUCCCAG 1593 54 CCACCAUGCCAAGUGGUCCCAGG 1594 55CACCAUGCCAAGUGGUCCCAGGC 1595 56 ACCAUGCCAAGUGGUCCCAGGCU 1596 57CCAUGCCAAGUGGUCCCAGGCUG 1597 58 CAUGCCAAGUGGUCCCAGGCUGC 1598 59AUGCCAAGUGGUCCCAGGCUGCA 1599 60 UGCCAAGUGGUCCCAGGCUGCAC 1600 61GCCAAGUGGUCCCAGGCUGCACC 1601 62 CCAAGUGGUCCCAGGCUGCACCC 1602 63CAAGUGGUCCCAGGCUGCACCCA 1603 64 AAGUGGUCCCAGGCUGCACCCAU 1604 65AGUGGUCCCAGGCUGCACCCAUG 1605 66 GUGGUCCCAGGCUGCACCCAUGG 1606 67UGGUCCCAGGCUGCACCCAUGGC 1607 68 GGUCCCAGGCUGCACCCAUGGCA 1608 69GUCCCAGGCUGCACCCAUGGCAG 1609 70 UCCCAGGCUGCACCCAUGGCAGA 1610 71CCCAGGCUGCACCCAUGGCAGAA 1611 72 CCAGGCUGCACCCAUGGCAGAAG 1612 73CAGGCUGCACCCAUGGCAGAAGG 1613 74 AGGCUGCACCCAUGGCAGAAGGA 1614 75GGCUGCACCCAUGGCAGAAGGAG 1615 76 GCUGCACCCAUGGCAGAAGGAGG 1616 77CUGCACCCAUGGCAGAAGGAGGA 1617 78 UGCACCCAUGGCAGAAGGAGGAG 1618 79GCACCCAUGGCAGAAGGAGGAGG 1619 80 CACCCAUGGCAGAAGGAGGAGGG 1620 81ACCCAUGGCAGAAGGAGGAGGGC 1621 82 CCCAUGGCAGAAGGAGGAGGGCA 1622 83CCAUGGCAGAAGGAGGAGGGCAG 1623 84 CAUGGCAGAAGGAGGAGGGCAGA 1624 85AUGGCAGAAGGAGGAGGGCAGAA 1625 86 UGGCAGAAGGAGGAGGGCAGAAU 1626 87GGCAGAAGGAGGAGGGCAGAAUC 1627 88 GCAGAAGGAGGAGGGCAGAAUCA 1628 89CAGAAGGAGGAGGGCAGAAUCAU 1629 90 AGAAGGAGGAGGGCAGAAUCAUC 1630 91GAAGGAGGAGGGCAGAAUCAUCA 1631 92 AAGGAGGAGGGCAGAAUCAUCAC 1632 93AGGAGGAGGGCAGAAUCAUCACG 1633 94 GGAGGAGGGCAGAAUCAUCACGA 1634 95GAGGAGGGCAGAAUCAUCACGAA 1635 96 AGGAGGGCAGAAUCAUCACGAAG 1636 97GGAGGGCAGAAUCAUCACGAAGU 1637 98 GAGGGCAGAAUCAUCACGAAGUG 1638 99AGGGCAGAAUCAUCACGAAGUGG 1639 100 GGGCAGAAUCAUCACGAAGUGGU 1640 101GGCAGAAUCAUCACGAAGUGGUG 1641 102 GCAGAAUCAUCACGAAGUGGUGA 1642 103CAGAAUCAUCACGAAGUGGUGAA 1643 104 AGAAUCAUCACGAAGUGGUGAAG 1644 105GAAUCAUCACGAAGUGGUGAAGU 1645 106 AAUCAUCACGAAGUGGUGAAGUU 1646 107AUCAUCACGAAGUGGUGAAGUUC 1647 108 UCAUCACGAAGUGGUGAAGUUCA 1648 109CAUCACGAAGUGGUGAAGUUCAU 1649 110 AUCACGAAGUGGUGAAGUUCAUG 1650 111UCACGAAGUGGUGAAGUUCAUGG 1651 112 CACGAAGUGGUGAAGUUCAUGGA 1652 113ACGAAGUGGUGAAGUUCAUGGAU 1653 114 CGAAGUGGUGAAGUUCAUGGAUG 1654 115GAAGUGGUGAAGUUCAUGGAUGU 1655 116 AAGUGGUGAAGUUCAUGGAUGUC 1656 117AGUGGUGAAGUUCAUGGAUGUCU 1657 118 GUGGUGAAGUUCAUGGAUGUCUA 1658 119UGGUGAAGUUCAUGGAUGUCUAU 1659 120 GGUGAAGUUCAUGGAUGUCUAUC 1660 121GUGAAGUUCAUGGAUGUCUAUCA 1661 122 UGAAGUUCAUGGAUGUCUAUCAG 1662 123GAAGUUCAUGGAUGUCUAUCAGC 1663 124 AAGUUCAUGGAUGUCUAUCAGCG 1664 125AGUUCAUGGAUGUCUAUCAGCGC 1665 126 GUUCAUGGAUGUCUAUCAGCGCA 1666 127UUCAUGGAUGUCUAUCAGCGCAG 1667 128 UCAUGGAUGUCUAUCAGCGCAGC 1668 129CAUGGAUGUCUAUCAGCGCAGCU 1669 130 AUGGAUGUCUAUCAGCGCAGCUA 1670 131UGGAUGUCUAUCAGCGCAGCUAC 1671 132 GGAUGUCUAUCAGCGCAGCUACU 1672 133GAUGUCUAUCAGCGCAGCUACUG 1673 134 AUGUCUAUCAGCGCAGCUACUGC 1674 135UGUCUAUCAGCGCAGCUACUGCC 1675 136 GUCUAUCAGCGCAGCUACUGCCA 1676 137UCUAUCAGCGCAGCUACUGCCAU 1677 138 CUAUCAGCGCAGCUACUGCCAUC 1678 139UAUCAGCGCAGCUACUGCCAUCC 1679 140 AUCAGCGCAGCUACUGCCAUCCA 1680 141UCAGCGCAGCUACUGCCAUCCAA 1681 142 CAGCGCAGCUACUGCCAUCCAAU 1682 143AGCGCAGCUACUGCCAUCCAAUC 1683 144 GCGCAGCUACUGCCAUCCAAUCG 1684 145CGCAGCUACUGCCAUCCAAUCGA 1685 146 GCAGCUACUGCCAUCCAAUCGAG 1686 147CAGCUACUGCCAUCCAAUCGAGA 1687 148 AGCUACUGCCAUCCAAUCGAGAC 1688 149GCUACUGCCAUCCAAUCGAGACC 1689 150 CUACUGCCAUCCAAUCGAGACCC 1690 151UACUGCCAUCCAAUCGAGACCCU 1691 152 ACUGCCAUCCAAUCGAGACCCUG 1692 153CUGCCAUCCAAUCGAGACCCUGG 1693 154 UGCCAUCCAAUCGAGACCCUGGU 1694 155GCCAUCCAAUCGAGACCCUGGUG 1695 156 CCAUCCAAUCGAGACCCUGGUGG 1696 157CAUCCAAUCGAGACCCUGGUGGA 1697 158 AUCCAAUCGAGACCCUGGUGGAC 1698 159UCCAAUCGAGACCCUGGUGGACA 1699 160 CCAAUCGAGACCCUGGUGGACAU 1700 161CAAUCGAGACCCUGGUGGACAUC 1701 162 AAUCGAGACCCUGGUGGACAUCU 1702 163AUCGAGACCCUGGUGGACAUCUU 1703 164 UCGAGACCCUGGUGGACAUCUUC 1704 165CGAGACCCUGGUGGACAUCUUCC 1705 166 GAGACCCUGGUGGACAUCUUCCA 1706 167AGACCCUGGUGGACAUCUUCCAG 1707 168 GACCCUGGUGGACAUCUUCCAGG 1708 169ACCCUGGUGGACAUCUUCCAGGA 1709 170 CCCUGGUGGACAUCUUCCAGGAG 1710 171CCUGGUGGACAUCUUCCAGGAGU 1711 172 CUGGUGGACAUCUUCCAGGAGUA 1712 173UGGUGGACAUCUUCCAGGAGUAC 1713 174 GGUGGACAUCUUCCAGGAGUACC 1714 175GUGGACAUCUUCCAGGAGUACCC 1715 176 UGGACAUCUUCCAGGAGUACCCU 1716 177GGACAUCUUCCAGGAGUACCCUG 1717 178 GACAUCUUCCAGGAGUACCCUGA 1718 179ACAUCUUCCAGGAGUACCCUGAU 1719 180 CAUCUUCCAGGAGUACCCUGAUG 1720 181AUCUUCCAGGAGUACCCUGAUGA 1721 182 UCUUCCAGGAGUACCCUGAUGAG 1722 183CUUCCAGGAGUACCCUGAUGAGA 1723 184 UUCCAGGAGUACCCUGAUGAGAU 1724 185UCCAGGAGUACCCUGAUGAGAUC 1725 186 CCAGGAGUACCCUGAUGAGAUCG 1726 187CAGGAGUACCCUGAUGAGAUCGA 1727 188 AGGAGUACCCUGAUGAGAUCGAG 1728 189GGAGUACCCUGAUGAGAUCGAGU 1729 190 GAGUACCCUGAUGAGAUCGAGUA 1730 191AGUACCCUGAUGAGAUCGAGUAC 1731 192 GUACCCUGAUGAGAUCGAGUACA 1732 193UACCCUGAUGAGAUCGAGUACAU 1733 194 ACCCUGAUGAGAUCGAGUACAUC 1734 195CCCUGAUGAGAUCGAGUACAUCU 1735 196 CCUGAUGAGAUCGAGUACAUCUU 1736 197CUGAUGAGAUCGAGUACAUCUUC 1737 198 UGAUGAGAUCGAGUACAUCUUCA 1738 199GAUGAGAUCGAGUACAUCUUCAA 1739 200 AUGAGAUCGAGUACAUCUUCAAG 1740 201UGAGAUCGAGUACAUCUUCAAGC 1741 202 GAGAUCGAGUACAUCUUCAAGCC 1742 203AGAUCGAGUACAUCUUCAAGCCA 1743 204 GAUCGAGUACAUCUUCAAGCCAU 1744 205AUCGAGUACAUCUUCAAGCCAUC 1745 206 UCGAGUACAUCUUCAAGCCAUCC 1746 207CGAGUACAUCUUCAAGCCAUCCU 1747 208 GAGUACAUCUUCAAGCCAUCCUG 1748 209AGUACAUCUUCAAGCCAUCCUGU 1749 210 GUACAUCUUCAAGCCAUCCUGUG 1750 211UACAUCUUCAAGCCAUCCUGUGU 1751 212 ACAUCUUCAAGCCAUCCUGUGUG 1752 213CAUCUUCAAGCCAUCCUGUGUGC 1753 214 AUCUUCAAGCCAUCCUGUGUGCC 1754 215UCUUCAAGCCAUCCUGUGUGCCC 1755 216 CUUCAAGCCAUCCUGUGUGCCCC 1756 217UUCAAGCCAUCCUGUGUGCCCCU 1757 218 UCAAGCCAUCCUGUGUGCCCCUG 1758 219CAAGCCAUCCUGUGUGCCCCUGA 1759 220 AAGCCAUCCUGUGUGCCCCUGAU 1760 221AGCCAUCCUGUGUGCCCCUGAUG 1761 222 GCCAUCCUGUGUGCCCCUGAUGC 1762 223CCAUCCUGUGUGCCCCUGAUGCG 1763 224 CAUCCUGUGUGCCCCUGAUGCGA 1764 225AUCCUGUGUGCCCCUGAUGCGAU 1765 226 UCCUGUGUGCCCCUGAUGCGAUG 1766 227CCUGUGUGCCCCUGAUGCGAUGC 1767 228 CUGUGUGCCCCUGAUGCGAUGCG 1768 229UGUGUGCCCCUGAUGCGAUGCGG 1769 230 GUGUGCCCCUGAUGCGAUGCGGG 1770 231UGUGCCCCUGAUGCGAUGCGGGG 1771 232 GUGCCCCUGAUGCGAUGCGGGGG 1772 233UGCCCCUGAUGCGAUGCGGGGGC 1773 234 GCCCCUGAUGCGAUGCGGGGGCU 1774 235CCCCUGAUGCGAUGCGGGGGCUG 1775 236 CCCUGAUGCGAUGCGGGGGCUGC 1776 237CCUGAUGCGAUGCGGGGGCUGCU 1777 238 CUGAUGCGAUGCGGGGGCUGCUG 1778 239UGAUGCGAUGCGGGGGCUGCUGC 1779 240 GAUGCGAUGCGGGGGCUGCUGCA 1780 241AUGCGAUGCGGGGGCUGCUGCAA 1781 242 UGCGAUGCGGGGGCUGCUGCAAU 1782 243GCGAUGCGGGGGCUGCUGCAAUG 1783 244 CGAUGCGGGGGCUGCUGCAAUGA 1784 245GAUGCGGGGGCUGCUGCAAUGAC 1785 246 AUGCGGGGGCUGCUGCAAUGACG 1786 247UGCGGGGGCUGCUGCAAUGACGA 1787 248 GCGGGGGCUGCUGCAAUGACGAG 1788 249CGGGGGCUGCUGCAAUGACGAGG 1789 250 GGGGGCUGCUGCAAUGACGAGGG 1790 251GGGGCUGCUGCAAUGACGAGGGC 1791 252 GGGCUGCUGCAAUGACGAGGGCC 1792 253GGCUGCUGCAAUGACGAGGGCCU 1793 254 GCUGCUGCAAUGACGAGGGCCUG 1794 255CUGCUGCAAUGACGAGGGCCUGG 1795 256 UGCUGCAAUGACGAGGGCCUGGA 1796 257GCUGCAAUGACGAGGGCCUGGAG 1797 258 CUGCAAUGACGAGGGCCUGGAGU 1798 259UGCAAUGACGAGGGCCUGGAGUG 1799 260 GCAAUGACGAGGGCCUGGAGUGU 1800 261CAAUGACGAGGGCCUGGAGUGUG 1801 262 AAUGACGAGGGCCUGGAGUGUGU 1802 263AUGACGAGGGCCUGGAGUGUGUG 1803 264 UGACGAGGGCCUGGAGUGUGUGC 1804 265GACGAGGGCCUGGAGUGUGUGCC 1805 266 ACGAGGGCCUGGAGUGUGUGCCC 1806 267CGAGGGCCUGGAGUGUGUGCCCA 1807 268 GAGGGCCUGGAGUGUGUGCCCAC 1808 269AGGGCCUGGAGUGUGUGCCCACU 1809 270 GGGCCUGGAGUGUGUGCCCACUG 1810 271GGCCUGGAGUGUGUGCCCACUGA 1811 272 GCCUGGAGUGUGUGCCCACUGAG 1812 273CCUGGAGUGUGUGCCCACUGAGG 1813 274 CUGGAGUGUGUGCCCACUGAGGA 1814 275UGGAGUGUGUGCCCACUGAGGAG 1815 276 GGAGUGUGUGCCCACUGAGGAGU 1816 277GAGUGUGUGCCCACUGAGGAGUC 1817 278 AGUGUGUGCCCACUGAGGAGUCC 1818 279GUGUGUGCCCACUGAGGAGUCCA 1819 280 UGUGUGCCCACUGAGGAGUCCAA 1820 281GUGUGCCCACUGAGGAGUCCAAC 1821 282 UGUGCCCACUGAGGAGUCCAACA 1822 283GUGCCCACUGAGGAGUCCAACAU 1823 284 UGCCCACUGAGGAGUCCAACAUC 1824 285GCCCACUGAGGAGUCCAACAUCA 1825 286 CCCACUGAGGAGUCCAACAUCAC 1826 287CCACUGAGGAGUCCAACAUCACC 1827 288 CACUGAGGAGUCCAACAUCACCA 1828 289ACUGAGGAGUCCAACAUCACCAU 1829 290 CUGAGGAGUCCAACAUCACCAUG 1830 291UGAGGAGUCCAACAUCACCAUGC 1831 292 GAGGAGUCCAACAUCACCAUGCA 1832 293AGGAGUCCAACAUCACCAUGCAG 1833 294 GGAGUCCAACAUCACCAUGCAGA 1834 295GAGUCCAACAUCACCAUGCAGAU 1835 296 AGUCCAACAUCACCAUGCAGAUU 1836 297GUCCAACAUCACCAUGCAGAUUA 1837 298 UCCAACAUCACCAUGCAGAUUAU 1838 299CCAACAUCACCAUGCAGAUUAUG 1839 300 CAACAUCACCAUGCAGAUUAUGC 1840 301AACAUCACCAUGCAGAUUAUGCG 1841 302 ACAUCACCAUGCAGAUUAUGCGG 1842 303CAUCACCAUGCAGAUUAUGCGGA 1843 304 AUCACCAUGCAGAUUAUGCGGAU 1844 305UCACCAUGCAGAUUAUGCGGAUC 1845 306 CACCAUGCAGAUUAUGCGGAUCA 1846 307ACCAUGCAGAUUAUGCGGAUCAA 1847 308 CCAUGCAGAUUAUGCGGAUCAAA 1848 309CAUGCAGAUUAUGCGGAUCAAAC 1849 310 AUGCAGAUUAUGCGGAUCAAACC 1850 311UGCAGAUUAUGCGGAUCAAACCU 1851 312 GCAGAUUAUGCGGAUCAAACCUC 1852 313CAGAUUAUGCGGAUCAAACCUCA 1853 314 AGAUUAUGCGGAUCAAACCUCAC 1854 315GAUUAUGCGGAUCAAACCUCACC 1855 316 AUUAUGCGGAUCAAACCUCACCA 1856 317UUAUGCGGAUCAAACCUCACCAA 1857 318 UAUGCGGAUCAAACCUCACCAAG 1858 319AUGCGGAUCAAACCUCACCAAGG 1859 320 UGCGGAUCAAACCUCACCAAGGC 1860 321GCGGAUCAAACCUCACCAAGGCC 1861 322 CGGAUCAAACCUCACCAAGGCCA 1862 323GGAUCAAACCUCACCAAGGCCAG 1863 324 GAUCAAACCUCACCAAGGCCAGC 1864 325AUCAAACCUCACCAAGGCCAGCA 1865 326 UCAAACCUCACCAAGGCCAGCAC 1866 327CAAACCUCACCAAGGCCAGCACA 1867 328 AAACCUCACCAAGGCCAGCACAU 1868 329AACCUCACCAAGGCCAGCACAUA 1869 330 ACCUCACCAAGGCCAGCACAUAG 1870 331CCUCACCAAGGCCAGCACAUAGG 1871 332 CUCACCAAGGCCAGCACAUAGGA 1872 333UCACCAAGGCCAGCACAUAGGAG 1873 334 CACCAAGGCCAGCACAUAGGAGA 1874 335ACCAAGGCCAGCACAUAGGAGAG 1875 336 CCAAGGCCAGCACAUAGGAGAGA 1876 337CAAGGCCAGCACAUAGGAGAGAU 1877 338 AAGGCCAGCACAUAGGAGAGAUG 1878 339AGGCCAGCACAUAGGAGAGAUGA 1879 340 GGCCAGCACAUAGGAGAGAUGAG 1880 341GCCAGCACAUAGGAGAGAUGAGC 1881 342 CCAGCACAUAGGAGAGAUGAGCU 1882 343CAGCACAUAGGAGAGAUGAGCUU 1883 344 AGCACAUAGGAGAGAUGAGCUUC 1884 345GCACAUAGGAGAGAUGAGCUUCC 1885 346 CACAUAGGAGAGAUGAGCUUCCU 1886 347ACAUAGGAGAGAUGAGCUUCCUA 1887 348 CAUAGGAGAGAUGAGCUUCCUAC 1888 349AUAGGAGAGAUGAGCUUCCUACA 1889 350 UAGGAGAGAUGAGCUUCCUACAG 1890 351AGGAGAGAUGAGCUUCCUACAGC 1891 352 GGAGAGAUGAGCUUCCUACAGCA 1892 353GAGAGAUGAGCUUCCUACAGCAC 1893 354 AGAGAUGAGCUUCCUACAGCACA 1894 355GAGAUGAGCUUCCUACAGCACAA 1895 356 AGAUGAGCUUCCUACAGCACAAC 1896 357GAUGAGCUUCCUACAGCACAACA 1897 358 AUGAGCUUCCUACAGCACAACAA 1898 359UGAGCUUCCUACAGCACAACAAA 1899 360 GAGCUUCCUACAGCACAACAAAU 1900 361AGCUUCCUACAGCACAACAAAUG 1901 362 GCUUCCUACAGCACAACAAAUGU 1902 363CUUCCUACAGCACAACAAAUGUG 1903 364 UUCCUACAGCACAACAAAUGUGA 1904 365UCCUACAGCACAACAAAUGUGAA 1905 366 CCUACAGCACAACAAAUGUGAAU 1906 367CUACAGCACAACAAAUGUGAAUG 1907 368 UACAGCACAACAAAUGUGAAUGC 1908 369ACAGCACAACAAAUGUGAAUGCA 1909 370 CAGCACAACAAAUGUGAAUGCAG 1910 371AGCACAACAAAUGUGAAUGCAGA 1911 372 GCACAACAAAUGUGAAUGCAGAC 1912 373CACAACAAAUGUGAAUGCAGACC 1913 374 ACAACAAAUGUGAAUGCAGACCA 1914 375CAACAAAUGUGAAUGCAGACCAA 1915 376 AACAAAUGUGAAUGCAGACCAAA 1916 377ACAAAUGUGAAUGCAGACCAAAG 1917 378 CAAAUGUGAAUGCAGACCAAAGA 1918 379AAAUGUGAAUGCAGACCAAAGAA 1919 380 AAUGUGAAUGCAGACCAAAGAAA 1920 381AUGUGAAUGCAGACCAAAGAAAG 1921 382 UGUGAAUGCAGACCAAAGAAAGA 1922 383GUGAAUGCAGACCAAAGAAAGAU 1923 384 UGAAUGCAGACCAAAGAAAGAUA 1924 385GAAUGCAGACCAAAGAAAGAUAG 1925 386 AAUGCAGACCAAAGAAAGAUAGA 1926 387AUGCAGACCAAAGAAAGAUAGAG 1927 388 UGCAGACCAAAGAAAGAUAGAGC 1928 389GCAGACCAAAGAAAGAUAGAGCA 1929 390 CAGACCAAAGAAAGAUAGAGCAA 1930 391AGACCAAAGAAAGAUAGAGCAAG 1931 392 GACCAAAGAAAGAUAGAGCAAGA 1932 393ACCAAAGAAAGAUAGAGCAAGAC 1933 394 CCAAAGAAAGAUAGAGCAAGACA 1934 395CAAAGAAAGAUAGAGCAAGACAA 1935 396 AAAGAAAGAUAGAGCAAGACAAG 1936 397AAGAAAGAUAGAGCAAGACAAGA 1937 398 AGAAAGAUAGAGCAAGACAAGAA 1938 399GAAAGAUAGAGCAAGACAAGAAA 1939 400 AAAGAUAGAGCAAGACAAGAAAA

TABLE 4b VEGF targeted duplexes position SEQ SEQ in ID Target sequenceID ORF NO: (5′-3′) Duplex ID Strand NO: Strand Sequences 1 2184AUGAACUUUCUGCUGUCUUGGGU AL-DP-4043 S 1940 5 GAACUUUCUGCUGUCUUGGGU 3 AS1941 3 UACUUGAAAGACGACAGAACCCA 5 22 2185 GUGCAUUGGAGCCUUGCCUUGCUAL-DP-4077 S 1942 5 GCAUUGGAGCCUUGCCUUGCU 3 AS 1943 3CACGUAACCUCGGAACGGAACGA 5 47 2186 UCUACCUCCACCAUGCCAAGUGG AL-DP-4021 S1944 5 UACCUCCACCAUGCCAAGUTT 3 AS 1945 3 TTAUGGAGGUGGUACGGUUCA 5 48 2187CUACCUCCACCAUGCCAAGUGGU AL-DP-4109 S 1946 5 ACCUCCACCAUGCCAAGUGTT 3 AS1947 3 TTUGGAGGUGGUACGGUUCAC 5 50 2188 ACCUCCACCAUGCCAAGUGGUCCAL-DP-4006 S 1948 5 CUCCACCAUGCCAAGUGGUCC 3 AS 1949 3UGGAGGUGGUACGGUUCACCAGG 5 AL-DP-4083 S 1950 5 CUCCACCAUGCCAAGUGGUTT 3 AS1951 3 TTGAGGUGGUACGGUUCACCA 5 51 2189 CCUCCACCAUGCCAAGUGGUCCCAL-DP-4047 S 1952 5 UCCACCAUGCCAAGUGGUCCC 3 AS 1953 3GGAGGUGGUACGGUUCACCAGGG 5 AL-DP-4017 S 1954 5 UCCACCAUGCCAAGUGGUCTT 3 AS1955 3 TTAGGUGGUACGGUUCACCAG 5 52 2190 CUCCACCAUGCCAAGUGGUCCCAAL-DP-4048 S 1956 5 CCACCAUGCCAAGUGGUCCCA 3 AS 1957 3GAGGUGGUACGGUUCACCAGGGU 5 AL-DP-4103 S 1958 5 CCACCAUGCCAAGUGGUCCTT 3 AS1959 3 TTGGUGGUACGGUUCACCAGG 5 53 2191 UCCACCAUGCCAAGUGGUCCCAGAL-DP-4035 S 1960 5 CACCAUGCCAAGUGGUCCCAG 3 AS 1961 3AGGUGGUACGGUUCACCAGGGUC 5 AL-DP-4018 S 1962 5 CACCAUGCCAAGUGGUCCCTT 3 AS1963 3 TTGUGGUACGGUUCACCAGGG 5 54 2192 CCACCAUGCCAAGUGGUCCCAGGAL-DP-4036 S 1964 5 ACCAUGCCAAGUGGUCCCAGG 3 AS 1965 3GGUGGUACGGUUCACCAGGGUCC 5 AL-DP-4084 S 1966 5 ACCAUGCCAAGUGGUCCCATT 3 AS1967 3 TTUGGUACGGUUCACCAGGGU 5 55 2193 CACCAUGCCAAGUGGUCCCAGGCAL-DP-4093 S 1968 5 CCAUGCCAAGUGGUCCCAGGC 3 AS 1969 3GUGGUACGGUUCACCAGGGUCCG 5 AL-DP-4085 S 1970 5 CCAUGCCAAGUGGUCCCAGTT 3 AS1971 3 TTGGUACGGUUCACCAGGGUC 5 56 2194 ACCAUGCCAAGUGGUCCCAGGCUAL-DP-4037 S 1972 5 CAUGCCAAGUGGUCCCAGGCU 3 AS 1973 3UGGUACGGUUCACCAGGGUCCGA 5 AL-DP-4054 S 1974 5 CAUGCCAAGUGGUCCCAGGTT 3 AS1975 3 TTGUACGGUUCACCAGGGUCC 5 57 2195 CCAUGCCAAGUGGUCCCAGGCUGAL-DP-4038 S 1976 5 AUGCCAAGUGGUCCCAGGCUG 3 AS 1977 3GGUACGGUUCACCAGGGUCCGAC 5 AL-DP-4086 S 1978 5 AUGCCAAGUGGUCCCAGGCTT 3 AS1979 3 TTUACGGUUCACCAGGGUCCG 5 58 2196 CAUGCCAAGUGGUCCCAGGCUGCAL-DP-4049 S 1980 5 UGCCAAGUGGUCCCAGGCUGC 3 AS 1981 3GUACGGUUCACCAGGGUCCGACG 5 AL-DP-4087 S 1982 5 UGCCAAGUGGUCCCAGGCUTT 3 AS1983 3 TTACGGUUCACCAGGGUCCGA 5 59 2197 AUGCCAAGUGGUCCCAGGCUGCAAL-DP-4001 S 1984 5 GCCAAGUGGUCCCAGGCUGCA 3 AS 1985 3UACGGUUCACCAGGGUCCGACGU 5 AL-DP-4052 A 1986 5 GCCAAGUGGUCCCAGGCUGTT 3 AS1987 3 TTCGGUUCACCAGGGUCCGAC 5 60 2198 UGCCAAGUGGUCCCAGGCUGCACAL-DP-4007 S 1988 5 CCAAGUGGUCCCAGGCUGCAC 3 AS 1989 3ACGGUUCACCAGGGUCCGACGUG 5 AL-DP-4088 S 1990 5 CCAAGUGGUCCCAGGCUGCTT 3 AS1991 3 TTGGUUCACCAGGGUCCGACG 5 61 2199 GCCAAGUGGUCCCAGGCUGCACCAL-DP-4070 S 1992 5 CAAGUGGUCCCAGGCUGCACC 3 AS 1993 3CGGUUCACCAGGGUCCGACGUGG 5 AL-DP-4055 S 1994 5 CAAGUGGUCCCAGGCUGCATT 3 AS1995 3 TTGUUCACCAGGGUCCGACGU 5 62 2200 CCAAGUGGUCCCAGGCUGCACCCAL-DP-4071 S 1996 5 AAGUGGUCCCAGGCUGCACCC 3 AS 1997 3GGUUCACCAGGGUCCGACGUGGG 5 AL-DP-4056 S 1998 5 AAGUGGUCCCAGGCUGCACTT 3 AS1999 3 TTUUCACCAGGGUCCGACGUG 5 63 2201 CAAGUGGUCCCAGGCUGCACCCAAL-DP-4072 S 2000 5 AGUGGUCCCAGGCUGCACCCA 3 AS 2001 3GUUCACCAGGGUCCGACGUGGGU 5 AL-DP-4057 S 2002 5 AGUGGUCCCAGGCUGCACCTT 3 AS2003 3 TTUCACCAGGGUCCGACGUGG 5 64 2202 AAGUGGUCCCAGGCUGCACCCAUAL-DP-4066 S 2004 5 GUGGUCCCAGGCUGCACCCTT 3 AS 2005 3TTCACCAGGGUCCGACGUGGG 5 99 2203 AGGGCAGAAUCAUCACGAAGUGG AL-DP-4022 S2006 5 GGCAGAAUCAUCACGAAGUTT 3 AS 2007 3 TTCCGUCUUAGUAGUGCUUCA 5 1002204 GGGCAGAAUCAUCACGAAGUGGU AL-DP-4023 S 2008 5 GCAGAAUCAUCACGAAGUGTT 3AS 2009 3 TTCGUCUUAGUAGUGCUUCAC 5 101 2205 GGCAGAAUCAUCACGAAGUGGUGAL-DP-4024 S 2010 5 CAGAAUCAUCACGAAGUGGTT 3 AS 2011 3TTGUCUUAGUAGUGCUUCACC 5 102 2206 GCAGAAUCAUCACGAAGUGGUGA AL-DP-4076 S2012 5 AGAAUCAUCACGAAGUGGUGA 3 AS 2013 3 CGUCUUAGUAGUGCUUCACCACU 5AL-DP-4019 S 2014 5 AGAAUCAUCACGAAGUGGUTT 3 AS 2015 3TTUCUUAGUAGUGCUUCACCA 5 103 2207 CAGAAUCAUCACGAAGUGGUGAA AL-DP-4025 S2016 5 GAAUCAUCACGAAGUGGUGTT 3 AS 2017 3 TTCUUAGUAGUGCUUCACCAC 5 1042208 AGAAUCAUCACGAAGUGGUGAAG AL-DP-4110 S 2018 5 AAUCAUCACGAAGUGGUGATT 3AS 2019 3 TTUUAGUAGUGCUUCACCACU 5 105 2209 GAAUCAUCACGAAGUGGUGAAGUAL-DP-4068 S 2020 5 AUCAUCACGAAGUGGUGAATT 3 AS 2021 3TTUAGUAGUGCUUCACCACUU 5 113 2210 ACGAAGUGGUGAAGUUCAUGGAU AL-DP-4078 S2022 5 GAAGUGGUGAAGUUCAUGGAU 3 AS 2023 3 UGCUUCACCACUUCAAGUACCUA 5 1212211 GUGAAGUUCAUGGAUGUCUAUCA AL-DP-4080 S 2024 5 GAAGUUCAUGGAUGUCUAUCA 3AS 2025 3 CACUUCAAGUACCUACAGAUAGU 5 129 2212 CAUGGAUGUCUAUCAGCGCAGCUAL-DP-4111 S 2026 5 UGGAUGUCUAUCAGCGCAGTT 3 AS 2027 3TTACCUACAGAUAGUCGCGUC 5 130 2213 AUGGAUGUCUAUCAGCGCAGCUA AL-DP-4041 S2028 5 GGAUGUCUAUCAGCGCAGCUA 3 AS 2029 3 UACCUACAGAUAGUCGCGUCGAU 5AL-DP-4062 S 2030 5 GGAUGUCUAUCAGCGCAGCTT 3 AS 2031 3TTCCUACAGAUAGUCGCGUCG 5 131 2214 UGGAUGUCUAUCAGCGCAGCUAC AL-DP-4069 S2032 5 GAUGUCUAUCAGCGCAGCUTT 3 AS 2033 3 TTCUACAGAUAGUCGCGUCGA 5 1322215 GGAUGUCUAUCAGCGCAGCUACU AL-DP-4112 S 2034 5 AUGUCUAUCAGCGCAGCUATT 3AS 2035 3 TTUACAGAUAGUCGCGUCGAU 5 133 2216 GAUGUCUAUCAGCGCAGCUACUGAL-DP-4026 S 2036 5 UGUCUAUCAGCGCAGCUACTT 3 AS 2037 3TTACAGAUAGUCGCGUCGAUG 5 134 2217 AUGUCUAUCAGCGCAGCUACUGC AL-DP-4095 S2038 5 GUCUAUCAGCGCAGCUACUGC 3 AS 2039 3 UACAGAUAGUCGCGUCGAUGACG 5AL-DP-4020 S 2040 5 GUCUAUCAGCGCAGCUACUTT 3 AS 2041 3TTCAGAUAGUCGCGUCGAUGA 5 135 2218 UGUCUAUCAGCGCAGCUACUGCC AL-DP-4027 S2042 5 UCUAUCAGCGCAGCUACUGTT 3 AS 2043 3 TTAGAUAGUCGCGUCGAUGAC 5 1442219 GCGCAGCUACUGCCAUCCAAUCG AL-DP-4081 S 2044 5 GCAGCUACUGCCAUCCAAUCG 3AS 2045 3 CGCGUCGAUGACGGUAGGUUAGC 5 146 2220 GCAGCUACUGCCAUCCAAUCGAGAL-DP-4098 S 2046 5 AGCUACUGCCAUCCAAUCGAG 3 AS 2047 3CGUCGAUGACGGUAGGUUAGCUC 5 149 2221 GCUACUGCCAUCCAAUCGAGACC AL-DP-4028 S2048 5 UACUGCCAUCCAAUCGAGATT 3 AS 2049 3 TTAUGACGGUAGGUUAGCUCU 5 1502222 CUACUGCCAUCCAAUCGAGACCC AL-DP-4029 S 2050 5 ACUGCCAUCCAAUCGAGACTT 3AS 2051 3 TTUGACGGUAGGUUAGCUCUG 5 151 2223 UACUGCCAUCCAAUCGAGACCCUAL-DP-4030 S 2052 5 CUGCCAUCCAAUCGAGACCTT 3 AS 2053 3TTGACGGUAGGUUAGCUCUGG 5 152 2224 ACUGCCAUCCAAUCGAGACCCUG AL-DP-4031 S2054 5 UGCCAUCCAAUCGAGACCCTT 3 AS 2055 3 TTACGGUAGGUUAGCUCUGGG 5 1662225 GAGACCCUGGUGGACAUCUUCCA AL-DP-4008 S 2056 5 GACCCUGGUGGACAUCUUCCA 3AS 2057 3 CUCUGGGACCACCUGUAGAAGGU 5 AL-DP-4058 S 2058 5GACCCUGGUGGACAUCUUCTT 3 AS 2059 3 TTCUGGGACCACCUGUAGAAG 5 167 2226AGACCCUGGUGGACAUCUUCCAG AL-DP-4009 S 2060 5 ACCCUGGUGGACAUCUUCCAG 3 AS2061 3 UCUGGGACCACCUGUAGAAGGUC 5 AL-DP-4059 S 2062 5ACCCUGGUGGACAUCUUCCTT 3 AS 2063 3 TTUGGGACCACCUGUAGAAGG 5 168 2227GACCCUGGUGGACAUCUUCCAGG AL-DP-4010 S 2064 5 CCCUGGUGGACAUCUUCCAGG 3 AS2065 3 CUGGGACCACCUGUAGAAGGUCC 5 AL-DP-4060 S 2066 5CCCUGGUGGACAUCUUCCATT 3 AS 2067 3 TTGGGACCACCUGUAGAAGGU 5 169 2228ACCCUGGUGGACAUCUUCCAGGA AL-DP-4073 S 2068 5 CCUGGUGGACAUCUUCCAGGA 3 AS2069 3 UGGGACCACCUGUAGAAGGUCCU 5 AL-DP-4104 S 2070 5CCUGGUGGACAUCUUCCAGTT 3 AS 2071 3 TTGGACCACCUGUAGAAGGUC 5 170 2229CCCUGGUGGACAUCUUCCAGGAG AL-DP-4011 S 2072 5 CUGGUGGACAUCUUCCAGGAG 3 AS2073 3 GGGACCACCUGUAGAAGGUCCUC 5 AL-DP-4089 S 2074 5CUGGUGGACAUCUUCCAGGTT 3 AS 2075 3 TTGACCACCUGUAGAAGGUCC 5 171 2230CCUGGUGGACAUCUUCCAGGAGU AL-DP-4074 S 2076 5 UGGUGGACAUCUUCCAGGAGU 3 AS2077 3 GGACCACCUGUAGAAGGUCCUCA 5 AL-DP-4090 S 2078 5UGGUGGACAUCUUCCAGGATT 3 AS 2079 3 TTACCACCUGUAGAAGGUCCU 5 172 2231CUGGUGGACAUCUUCCAGGAGUA AL-DP-4039 S 2080 5 GGUGGACAUCUUCCAGGAGUA 3 AS2081 3 GACCACCUGUAGAAGGUCCUCAU 5 AL-DP-4091 S 2082 5GGUGGACAUCUUCCAGGAGTT 3 AS 2083 3 TTCCACCUGUAGAAGGUCCUC 5 175 2232GUGGACAUCUUCCAGGAGUACCC AL-DP-4003 S 2084 5 GGACAUCUUCCAGGAGUACCC 3 AS2085 3 CCUGUAGAAGGUCCUCAUGGG 5 AL-DP-4116 S 2086 5 GGACAUCUUCCAGGAGUACCC3 AS 2087 3 CCUGUAGAAGGUCCUCAUGGG 5 AL-DP-4015 S 2088 5GGACAUCUUCCAGGAGUACTT 3 AS 2089 3 TTCCUGUAGAAGGUCCUCAUG 5 AL-DP-4120 S2090 5 GGACAUCUUCCAGGAGUAC 3 AS 2091 3 CCUGUAGAAGGUCCUCAUG 5 179 2233ACAUCUUCCAGGAGUACCCUGAU AL-DP-4099 S 2092 5 AUCUUCCAGGAGUACCCUGAU 3 AS2093 3 UGUAGAAGGUCCUCAUGGGACUA 5 191 2234 AGUACCCUGAUGAGAUCGAGUACAL-DP-4032 S 2094 5 UACCCUGAUGAGAUCGAGUTT 3 AS 2095 3TTAUGGGACUACUCUAGCUCA 5 192 2235 GUACCCUGAUGAGAUCGAGUACA AL-DP-4042 S2096 5 ACCCUGAUGAGAUCGAGUACA 3 AS 2097 3 CAUGGGACUACUCUAGCUCAUGU 5AL-DP-4063 S 2098 5 ACCCUGAUGAGAUCGAGUATT 3 AS 2099 3TTUGGGACUACUCUAGCUCAU 5 209 2236 AGUACAUCUUCAAGCCAUCCUGU AL-DP-4064 S2100 5 UACAUCUUCAAGCCAUCCUTT 3 AS 2101 3 TTAUGUAGAAGUUCGGUAGGA 5 2602237 GCAAUGACGAGGGCCUGGAGUGU AL-DP-4044 S 2102 5 AAUGACGAGGGCCUGGAGUGU 3AS 2103 3 CGUUACUGCUCCCGGACCUCACA 5 263 2238 AUGACGAGGGCCUGGAGUGUGUGAL-DP-4045 S 2104 5 GACGAGGGCCUGGAGUGUGUG 3 AS 2105 3UACUGCUCCCGGACCUCACACAC 5 279 2239 GUGUGUGCCCACUGAGGAGUCCA AL-DP-4046 S2106 5 GUGUGCCCACUGAGGAGUCCA 3 AS 2107 3 CACACACGGGUGACUCCUCAGGU 5 2812240 GUGUGCCCACUGAGGAGUCCAAC AL-DP-4096 S 2108 5 GUGCCCACUGAGGAGUCCAAC 3AS 2109 3 CACACGGGUGACUCCUCAGGUUG 5 283 2241 GUGCCCACUGAGGAGUCCAACAUAL-DP-4040 S 2110 5 GCCCACUGAGGAGUCCAACAU 3 AS 2111 3CACGGGUGACUCCUCAGGUUGUA 5 289 2242 ACUGAGGAGUCCAACAUCACCAU AL-DP-4065 S2112 5 UGAGGAGUCCAACAUCACCTT 3 AS 2113 3 TTACUCCUCAGGUUGUAGUGG 5 3022243 ACAUCACCAUGCAGAUUAUGCGG AL-DP-4100 S 2114 5 AUCACCAUGCAGAUUAUGCGG 3AS 2115 3 UGUAGUGGUACGUCUAAUACGCC 5 305 2244 UCACCAUGCAGAUUAUGCGGAUCAL-DP-4033 S 2116 5 ACCAUGCAGAUUAUGCGGATT 3 AS 2117 3TTUGGUACGUCUAAUACGCCU 5 310 2245 AUGCAGAUUAUGCGGAUCAAACC AL-DP-4101 S2118 5 GCAGAUUAUGCGGAUCAAACC 3 AS 2119 3 UACGUCUAAUACGCCUAGUUUGG 5 3122246 GCAGAUUAUGCGGAUCAAACCUC AL-DP-4102 S 2120 5 AGAUUAUGCGGAUCAAACCUC 3AS 2121 3 CGUCUAAUACGCCUAGUUUGGAG 5 315 2247 GAUUAUGCGGAUCAAACCUCACCAL-DP-4034 S 2122 5 UUAUGCGGAUCAAACCUCATT 3 AS 2123 3TTAAUACGCCUAGUUUGGAGU 5 316 2248 AUUAUGCGGAUCAAACCUCACCA AL-DP-4113 S2124 5 UAUGCGGAUCAAACCUCACTT 3 AS 2125 3 TTAUACGCCUAGUUUGGAGUG 5 3172249 UUAUGCGGAUCAAACCUCACCAA AL-DP-4114 S 2126 5 AUGCGGAUCAAACCUCACCTT 3AS 2127 3 TTUACGCCUAGUUUGGAGUGG 5 319 2250 AUGCGGAUCAAACCUCACCAAGGAL-DP-4002 S 2128 5 GCGGAUCAAACCUCACCAAGG 3 AS 2129 3UACGCCUAGUUUGGAGUGGUUCC 5 AL-DP-4115 S 2130 5 GCGGAUCAAACCUCACCAA 3 AS2131 3 CGCCUAGUUUGGAGUGGUU 5 AL-DP-4014 S 2132 5 GCGGAUCAAACCUCACCAATT 3AS 2133 3 TTCGCCUAGUUUGGAGUGGUU 5 AL-DP-4119 S 2134 5GCGGAUCAAACCUCACCAA 3 AS 2135 3 CGCCUAGUUUGGAGUGGUU 5 321 2251GCGGAUCAAACCUCACCAAGGCC AL-DP-4013 S 2136 5 GGAUCAAACCUCACCAAGGCC 3 AS2137 3 CGCCUAGUUUGGAGUGGUUCCGG 5 341 2252 GCCAGCACAUAGGAGAGAUGAGCAL-DP-4075 S 2138 5 CAGCACAUAGGAGAGAUGAGC 3 AS 2139 3CGGUCGUGUAUCCUCUCUACUCG 5 AL-DP-4105 S 2140 5 CAGCACAUAGGAGAGAUGATT 3 AS2141 3 TTGUCGUGUAUCCUCUCUACU 5 342 2253 CCAGCACAUAGGAGAGAUGAGCUAL-DP-4050 S 2142 5 AGCACAUAGGAGAGAUGAGCU 3 AS 2143 3GGUCGUGUAUCCUCUCUACUCGA 5 AL-DP-4106 S 2144 5 AGCACAUAGGAGAGAUGAGTT 3 AS2145 3 TTUCGUGUAUCCUCUCUACUC 5 343 2254 CAGCACAUAGGAGAGAUGAGCUUAL-DP-4094 S 2146 5 GCACAUAGGAGAGAUGAGCUU 3 AS 2147 3GUCGUGUAUCCUCUCUACUCGAA 5 AL-DP-4118 S 2148 5 GCACAUAGGAGAGAUGAGCUU 3 AS2149 3 CGUGUAUCCUCUCUACUCGAA 5 AL-DP-4107 S 2150 5 GCACAUAGGAGAGAUGAGCTT3 AS 2151 3 TTCGUGUAUCCUCUCUACUCG 5 AL-DP-4122 S 2152 5GCACAUAGGAGAGAUGAGC 3 AS 2153 3 CGUGUAUCCUCUCUACUCG 5 344 2255AGCACAUAGGAGAGAUGAGCUUC AL-DP-4012 S 2154 5 CACAUAGGAGAGAUGAGCUUC 3 AS2155 3 UCGUGUAUCCUCUCUACUCGAAG 5 AL-DP-4108 S 2156 5CACAUAGGAGAGAUGAGCUTT 3 AS 2157 3 TTGUGUAUCCUCUCUACUCGA 5 346 2256CACAUAGGAGAGAUGAGCUUCCU AL-DP-4051 S 2158 5 CAUAGGAGAGAUGAGCUUCCU 3 AS2159 3 GUGUAUCCUCUCUACUCGAAGGA 5 AL-DP-4061 S 2160 5CAUAGGAGAGAUGAGCUUCTT 3 AS 2161 3 TTGUAUCCUCUCUACUCGAAG 5 349 2257AUAGGAGAGAUGAGCUUCCUACA AL-DP-4082 S 2162 5 AGGAGAGAUGAGCUUCCUACA 3 AS2163 3 UAUCCUCUCUACUCGAAGGAUGU 5 369 2258 ACAGCACAACAAAUGUGAAUGCAAL-DP-4079 S 2164 5 AGCACAACAAAUGUGAAUGCA 3 AS 2165 3UGUCGUGUUGUUUACACUUACGU 5 372 2259 GCACAACAAAUGUGAAUGCAGAC AL-DP-4097 S2166 5 ACAACAAAUGUGAAUGCAGAC 3 AS 2167 3 CGUGUUGUUUACACUUACGUCUG 5 3792260 AAAUGUGAAUGCAGACCAAAGAA AL-DP-4067 S 2168 5 AUGUGAAUGCAGACCAAAGTT 3AS 2169 3 TTUACACUUACGUCUGGUUUC 5 380 2261 AAUGUGAAUGCAGACCAAAGAAAAL-DP-4092 S 2170 5 UGUGAAUGCAGACCAAAGATT 3 AS 2171 3TTACACUUACGUCUGGUUUCU 5 381 2262 AUGUGAAUGCAGACCAAAGAAAG AL-DP-4004 S2172 5 GUGAAUGCAGACCAAAGAAAG 3 AS 2173 3 UACACUUACGUCUGGUUUCUUUC 5AL-DP-4117 S 2174 5 GUGAAUGCAGACCAAAGAAAG 3 AS 2175 3CACUUACGUCUGGUUUCUUUC 5 AL-DP-4016 S 2176 5 GUGAAUGCAGACCAAAGAATT 3 AS2177 3 TTCACUUACGUCUGGUUUCUU 5 AL-DP-4121 S 2178 5 GUGAAUGCAGACCAAAGAA 3AS 2179 3 CACUUACGUCUGGUUUCUU 5 383 2263 GUGAAUGCAGACCAAAGAAAGAUAL-DP-4005 S 2180 5 GAAUGCAGACCAAAGAAAGAU 3 AS 2181 3CACUUACGUCUGGUUUCUUUCUA 5 AL-DP-4053 S 2182 5 GAAUGCAGACCAAAGAAAGTT 3 AS2183 3 TTCUUACGUCUGGUUUCUUUC 5 Strand: S = sense, AS = Antisense

Example 2 Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, etal [2002] Biotechniques 33: 1244-8), a cell viability assay was used forsiRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3]or 10000 per well [Screen2])) were seeded in 96-well plates andsimultaneously transfected with Lipofectamine 2000 (Invitrogen) at afinal siRNA concentration in the well of 30 nM and at finalconcentrations of 50 nM (1^(st) screen) and 25 nM (2^(nd) screen). Asubset of duplexes was tested at 25 nM in a third screen (Table 5).

Seventy-two hours post-transfection, cell proliferation was assayed theaddition of WST-1 reagent (Roche) to the culture medium, and subsequentabsorbance measurement at 450 nm. The absorbance value for control(non-transfected) cells was considered 100 percent, and absorbances forthe siRNA transfected wells were compared to the control value. Assayswere performed in sextuplicate for each of three screens. A subset ofthe siRNAs was further tested at a range of siRNA concentrations. Assayswere performed in HeLa cells (14000 per well; method same as above,Table 5).

TABLE 5 Effects of Eg5 targeted duplexes on cell viability at 25 nM.Relative absorbance at 450 nm Screen I Screen II Screen III Duplex meansd Mean sd mean Sd AL-DP-6226 20 10 28 11 43 9 AL-DP-6227 66 27 96 41108 33 AL-DP-6228 56 28 76 22 78 18 AL-DP-6229 17 3 31 9 48 13AL-DP-6230 48 8 75 11 73 7 AL-DP-6231 8 1 21 4 41 10 AL-DP-6232 16 2 377 52 14 AL-DP-6233 31 9 37 6 49 12 AL-DP-6234 103 40 141 29 164 45AL-DP-6235 107 34 140 27 195 75 AL-DP-6236 48 12 54 12 56 12 AL-DP-623773 14 108 18 154 37 AL-DP-6238 64 9 103 10 105 24 AL-DP-6239 9 1 20 4 3111 AL-DP-6240 99 7 139 16 194 43 AL-DP-6241 43 9 54 12 66 19 AL-DP-62426 1 15 7 36 8 AL-DP-6243 7 2 19 5 33 13 AL-DP-6244 7 2 19 3 37 13AL-DP-6245 25 4 45 10 58 9 AL-DP-6246 34 8 65 10 66 13 AL-DP-6247 53 678 14 105 20 AL-DP-6248 7 0 22 7 39 12 AL-DP-6249 36 8 48 13 61 7

The nine siRNA duplexes that showed the greatest growth inhibition inTable 5 were re-tested at a range of siRNA concentrations in HeLa cells.The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM,1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed insextuplicate, and the concentration of each siRNA resulting in fiftypercent inhibition of cell proliferation (IC₅₀) was calculated. Thisdose-response analysis was performed between two and four times for eachduplex. Mean IC₅₀ values (nM) are given in Table 6.

TABLE 6 IC50 of siRNA: cell proliferation in HeLa cells Duplex Mean IC₅₀AL-DP-6226 15.5 AL-DP-6229 3.4 AL-DP-6231 4.2 AL-DP-6232 17.5 AL-DP-62394.4 AL-DP-6242 5.2 AL-DP-6243 2.6 AL-DP-6244 8.3 AL-DP-6248 1.9

Example 3 Eg5 siRNA In Vitro Screening Via mRNA Inhibition

Directly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCGPromochem GmbH, Wesel, Germany) cells were seeded at 1.5×10⁴ cells/wellon 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75μl of growth medium (Ham's F12, 10% fetal calf serum, 100 upenicillin/100 μg/ml streptomycin, all from Bookroom AG, Berlin,Germany). Transfections were performed in quadruplicates. For each well0.5 μl Lipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) weremixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at roomtemperature. For the siRNA concentration being 50 nM in the 100 μltransfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μlOpti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixtureand again incubated for 15 minutes at room temperature.siRNA-Lipofectamine2000-complexes were applied completely (25 μl eachper well) to the cells and cells were incubated for 24 h at 37° C. and5% CO₂ in a humidified incubator (Heroes GmbH, Hanau). The single dosescreen was done once at 50 nM and at 25 nM, respectively.

Cells were harvested by applying 50 μl of lysis mixture (content of theQuantiGene bDNA-kit from Genospectra, Fremont, USA) to each wellcontaining 100 μl of growth medium and were lysed at 53° C. for 30 min.Afterwards, 50 μl of the lists were incubated with probesets specific tohuman Eg5 and human GAPDH and proceeded according to the manufacturer'sprotocol for QuantiGene. In the end chemoluminescence was measured in aVictor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative lightunits) and values obtained with the hEg5 probeset were normalized to therespective GAPDH values for each well. Values obtained with siRNAsdirected against Eg5 were related to the value obtained with anunspecific siRNA (directed against HCV) which was set to 100% (Tables1b, 2b and 3b).

Effective siRNAs from the screen were further characterized by doseresponse curves. Transfections of dose response curves were performed atthe following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (nosiRNA) and diluted with Opti-MEM to a final concentration of 12.5 μlaccording to the above protocol. Data analysis was performed by usingthe Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey,UK) and applying the dose response model number 205 (Tables 1b, 2b and3b).

The lead siRNA AD12115 was additionally analyzed by applying theWST-proliferation assay from Roche (as previously described).

A subset of 34 duplexes from Table 2 that showed greatest activity wasassayed by transfection in HeLa cells at final concentrations rangingfrom 100 nM to 10fM. Transfections were performed in quadruplicate. Twodose-response assays were performed for each duplex. The concentrationgiving 20% (IC20), 50% (IC50) and 80% (IC80) reduction of KSP mRNA wascalculated for each duplex (Table 7).

TABLE 7 Dose response mRNA inhibition of Eg5/KSP duplexes in HeLa cellsConcentrations given in pM IC20s IC50s IC80s 1^(st) 2^(nd) 1st 2nd 1st2nd Duplex name screen screen screen screen screen screen AD12077 1.190.80 6.14 10.16 38.63 76.16 AD12078 25.43 25.43 156.18 156.18 ND NDAD12085 9.08 1.24 40.57 8.52 257.68 81.26 AD12095 1.03 0.97 9.84 4.9490.31 60.47 AD12113 4.00 5.94 17.18 28.14 490.83 441.30 AD12115 0.600.41 3.79 3.39 23.45 23.45 AD12125 31.21 22.02 184.28 166.15 896.851008.11 AD12134 2.59 5.51 17.87 22.00 116.36 107.03 AD12149 0.72 0.504.51 3.91 30.29 40.89 AD12151 0.53 6.84 4.27 10.72 22.88 43.01 AD12152155.45 7.56 867.36 66.69 13165.27 ND AD12157 0.30 26.23 14.60 92.0814399.22 693.31 AD12166 0.20 0.93 3.71 3.86 46.28 20.59 AD12180 28.8528.85 101.06 101.06 847.21 847.21 AD12185 2.60 0.42 15.55 13.91 109.80120.63 AD12194 2.08 1.11 5.37 5.09 53.03 30.92 AD12211 5.27 4.52 11.7318.93 26.74 191.07 AD12257 4.56 5.20 21.68 22.75 124.69 135.82 AD122802.37 4.53 6.89 20.23 64.80 104.82 AD12281 8.81 8.65 19.68 42.89 119.01356.08 AD12282 7.71 456.42 20.09 558.00 ND ND AD12285 ND 1.28 57.30 7.31261.79 42.53 AD12292 40.23 12.00 929.11 109.10 ND ND AD12252 0.02 18.636.35 68.24 138.09 404.91 AD12275 25.76 25.04 123.89 133.10 1054.54776.25 AD12266 4.85 7.80 10.00 32.94 41.67 162.65 AD12267 1.39 1.2112.00 4.67 283.03 51.12 AD12264 0.92 2.07 8.56 15.12 56.36 196.78AD12268 2.29 3.67 22.16 25.64 258.27 150.84 AD12279 1.11 28.54 23.1996.87 327.28 607.27 AD12256 7.20 33.52 46.49 138.04 775.54 1076.76AD12259 2.16 8.31 8.96 40.12 50.05 219.42 AD12276 19.49 6.14 89.60 59.60672.51 736.72 AD12321 4.67 4.91 24.88 19.43 139.50 89.49 (ND—notdetermined)

Example 4 Silencing of Liver Eg5/KSP in Juvenile Rats FollowingSingle-Bolus Administration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can bedetected in the growing rat liver. Target silencing with a formulatedEg5/KSP siRNA was evaluated in juvenile rats using duplex AD-6248.

KSP Duplex Tested

Duplex ID Target Sense Antisense AD6248 KSP AccGAAGuGuuGuuuGuccTsT (SEQID NO: 1238) GGAcAAAcAAcACUUCGGUTsT (SEQ ID NO: 1239)

Methods

Dosing of animals. Male, juvenile Sprague-Dawley rats (19 days old) wereadministered single doses of lipidoid (“LNP01”) formulated siRNA viatail vein injection. Groups of ten animals received doses of 10milligrams per kilogram (mg/kg) bodyweight of either AD6248 or anunspecific siRNA. Dose level refers to the amount of siRNA duplexadministered in the formulation. A third group receivedphosphate-buffered saline. Animals were sacrificed two days after siRNAadministration. Livers were dissected, flash frozen in liquid Nitrogenand pulverized into powders.

mRNA measurements. Levels of Eg5/KSP mRNA were measured in livers fromall treatment groups. Samples of each liver powder (approximately tenmilligrams) were homogenized in tissue lysis buffer containingproteinase K. Levels of Eg5/KSP and GAPDH mRNA were measured intriplicate for each sample using the Quantigene branched DNA assay(GenoSpectra). Mean values for Eg5/KSP were normalized to mean GAPDHvalues for each sample. Group means were determined and normalized tothe PBS group for each experiment.

Statistical analysis. Significance was determined by ANOVA followed bythe Tukey post-hoc test.

Results

Data Summary

Mean values (±standard deviation) for Eg5/KSP mRNA are given.Statistical significance (p value) versus the PBS group is shown (ns,not significant [p>0.05]).

TABLE 8 Experiment 1 KSP/GAPDH p value PBS 1.0 ± 0.47 AD6248 10 mg/kg0.47 ± 0.12  <0.001 unspec 10 mg/kg 1.0 ± 0.26 ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtainedfollowing treatment with formulated AD6248 at a dose of 10 mg/kg.

Example 5 Silencing of Rat Liver VEGF Following Intravenous Infusion ofLNP01 Formulated VSP

A “lipidoid” formulation comprising an equimolar mixture of two siRNAswas administered to rats. As used herein, VSP refers to a compositionhaving two siRNAs, one directed to Eg5/KSP and one directed to VEGF. Forthis experiment the duplex AD3133 directed towards VEGF and AD12115directed towards Eg5/KSP were used. Since Eg5/KSP expression is nearlyundetectable in the adult rat liver, only VEGF levels were measuredfollowing siRNA treatment.

siRNA duplexes administered (VSP) Duplex ID Target Sense AntisenseAD12115 Eg5/KSP ucGAGAAucuAAAcuAAcuTsT AGUuAGUUuAGAUUCUCGATsT (SEQ IDNO: 1240) (SEQ ID NO: 1241) AD3133 VEGF GcAcAuAGGAGAGAuGAGCUsUAAGCUcAUCUCUCCuAuGuGCusG (SEQ ID NO: 1242) (SEQ ID NO: 1243) Key:A,G,C,U-ribonucleotides; c,u-2′-O-Me ribonucleotides;s-phosphorothioate.

Unmodified versions of each strand and the targets for each siRNA are asfollows

Eg5/KSP unmod sense 5′ UCGAGAAUCUAAACUAACUTT 3′ SEQ ID NO: 1534 unmodantisense 3′ TTAGUCCUUAGAUUUGAUUGA 5′ SEQ ID NO: 1535 target5′ UCGAGAAUCUAAACUAACU 3′ SEQ ID NO: 1311 VEGF unmod sense5′ GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 1536 unmod antisense3′ GUCGUGUAUCCUCUCUACUCGAA 5′ SEQ ID NO: 1537 target5′ GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 1538

Methods

Dosing of animals. Adult, female Sprague-Dawley rats were administeredlipidoid (“LNP01”) formulated siRNA by a two-hour infusion into thefemoral vein. Groups of four animals received doses of 5, 10 and 15milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Doselevel refers to the total amount of siRNA duplex administered in theformulation. A fourth group received phosphate-buffered saline. Animalswere sacrificed 72 hours after the end of the siRNA infusion. Liverswere dissected, flash frozen in liquid Nitrogen and pulverized intopowders.

Formulation Procedure

The lipidoid ND98·4HCl (MW 1487) (Formula 1, above), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used toprepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16,100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions werethen combined in a 42:48:10 molar ratio. Combined lipid solution wasmixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that thefinal ethanol concentration was 35-45% and the final sodium acetateconcentration was 100-300 mM. Lipid-siRNA nanoparticles formedspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%. For SNALPformulation, the particle size is at least 30 nm, at least 40 nm, atleast 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferredrange is about at least 50 nm to about at least 110 nm, preferably aboutat least 60 nm to about at least 100 nm, most preferably about at least80 nm to about at least 90 nm. In one example, each of the particle sizecomprises at least about 1:1 ratio of Eg5 dsRNA to VEGF dsRNA.

mRNA measurements. Samples of each liver powder (approximately tenmilligrams) were homogenized in tissue lysis buffer containingproteinase K. Levels of VEGF and GAPDH mRNA were measured in triplicatefor each sample using the Quantigene branched DNA assay (GenoSpectra).Mean values for VEGF were normalized to mean GAPDH values for eachsample. Group means were determined and normalized to the PBS group foreach experiment.

Protein measurements. Samples of each liver powder (approximately 60milligrams) were homogenized in 1 ml RIPA buffer. Total proteinconcentrations were determined using the Micro BCA protein assay kit(Pierce). Samples of total protein from each animal was used todetermine VEGF protein levels using a VEGF ELISA assay (R&D systems).Group means were determined and normalized to the PBS group for eachexperiment.

Statistical analysis. Significance was determined by ANOVA followed bythe Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein(rel. VEGF) are shown for each treatment group. Statistical significance(p value) versus the PBS group for each experiment is shown.

TABLE 9 VEGF/GAPDH p value rel VEGF p value PBS  1.0 ± 0.17  1.0 ± 0.17 5 mg/kg 0.74 ± 0.12 <0.05 0.23 ± 0.03 <0.001 10 mg/kg 0.65 ± 0.12<0.005 0.22 ± 0.03 <0.001 15 mg/kg 0.49 ± 0.17 <0.001 0.20 ± 0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein weremeasured at all three siRNA dose levels.

Example 6 Assessment of VSP SNALP in Mouse Models of Human HepaticTumors

These studies utilized a VSP siRNA cocktail containing dsRNAs targetingKSP/Eg5 and dsRNAs targeting VEGF. As used herein, VSP refers to acomposition having two siRNAs, one directed to Eg5/KSP and one directedto VEGF. For this experiment the duplexes AD3133 (directed towards VEGF)and AD12115 (directed towards Eg5/KSP) were used. The siRNA cocktail wasformulated in SNALPs.

The maximum study size utilized 20-25 mice. To test the efficacy of thesiRNA SNALP cocktail to treat liver cancer, 1×10̂6 tumor cells wereinjected directly into the left lateral lobe of test mice. The incisionswere closed by sutures, and the mice allowed to recover for 2-5 hours.The mice were fully recovered within 48-72 hours. The SNALP siRNAtreatment was initiated 8-11 days after tumor seeding.

The SNALP formulations utilized were (i) VSP (KSP+VEGF siRNA cocktail(1:1 molar ratio)); (ii) KSP (KSP+Luc siRNA cocktail); and (iii) VEGF(VEGF+Luc siRNA cocktail). All formulations contained equal amounts (mg)of each active siRNA. All mice received a total siRNA/lipid dose, andeach cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1%DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug usingoriginal citrate buffer conditions.

Human Hep3B Study A: Anti-Tumor Activity of VSP-SNALP

Human Hepatoma Hep3B tumors were established in scid/beige mice byintrahepatic seeding. Group A (n=6) animals were administered PBS; GroupB (n=6) animals were administered VSP SNALP; Group C (n=5) animals wereadministered KSP/Luc SNALP; and Group D (n=5) animals were administeredVEGF/Luc SNALP.

SNALP treatment was initiated eight days after tumor seeding. The SNALPwas dosed at 3 mg/kg total siRNA, twice weekly (Monday and Thursday),for a total of six doses (cumulative 18 mg/kg siRNA). The final dose wasadministered at day 25, and the terminal endpoint was at day 27.

Tumor burden was assayed by (a) body weight; (b) liver weight; (c)visual inspection+photography at day 27; (d) human-specific mRNAanalysis; and (e) blood alpha-fetoprotein levels measured at day 27.

Table 10 below illustrates the results of visual scoring of tumor burdenmeasured in the seeded (left lateral) liver lobe. Score: “−”=no visibletumor; “+”=evidence of tumor tissue at injection site; “++”=Discretetumor nodule protruding from liver lobe; “+++”=large tumor protruding onboth sides of liver lobe; “++++”=large tumor, multiple nodulesthroughout liver lobe.

TABLE 10 Mouse Tumor Burden Group A: PBS, day 27 1 ++++ 2 ++++ 3 ++ 4+++ 5 ++++ 6 ++++ Group B: VSP 1 + (VEGF + KSP/Eg5, d. 27 2 − 3 − 4 − 5++ 6 − Group C: KSP 1 + (Luc + KSP), d. 27 2 ++ 3 − 4 + 5 ++ Group D:VEGF 1 ++++ (Luc + VEGF), d. 27 2 − 3 ++++ 4 +++ 5 ++++

Liver weights, as percentage of body weight, are shown in FIG. 1.

Body weights are shown in FIGS. 2A-2D.

From this study, the following conclusions were made. (1) VSP SNALPdemonstrated potent anti-tumor effects in Hep3B1H model; (2) theanti-tumor activity of the VSP cocktail appeared largely associated withthe KSP component; (3) anti-KSP activity was confirmed by single dosehistological analysis; and (4) VEGF siRNA showed no measurable effect oninhibition of tumor growth in this model.

Human Hep3B Study B: Prolonged Survival with VSP Treatment

In a second Hep3B study, human hepatoma Hep3B tumors were established byintrahepatic seeding into scid/beige mice. These mice were deficient forlymphocytes and natural killer (NK) cells, which is the minimal scopefor immune-mediated anti-tumor effects. Group A (n=6) mice wereuntreated; Group B (n=6) mice were administered luciferase (luc) 1955SNALP (Lot No. AP10-02); and Group C (n=7) mice were administered VSPSNALP (Lot No. AP10-01). SNALP was 1:57 cDMA SNALP, and 6:1 lipid:drug.

SNALP treatment was initiated eight days after tumor seeding. SNALP wasdosed at 3 mg/kg siRNA, twice weekly (Mondays and Thursdays), for atotal of six doses (cumulative 18 mg/kg siRNA). The final dose wasdelivered at day 25, and the terminal endpoint of the study was at day27.

Tumor burden was assayed by (1) body weight; (2) visualinspection+photography at day 27; (3) human-specific mRNA analysis; and(4) blood alpha-fetoprotein measured at day 27.

Body weights were measured at each day of dosing (days 8, 11, 14, 18,21, and 25) and on the day of sacrifice (FIG. 3).

TABLE 11 Tumor Burden by macroscopic Mouse observation Group A:untreated, A1R ++ day 27 A1G ++++ A1W − A2R ++++ A2G +++ A2W ++++ GroupB: B1R ++++ 1955 Luc SNALP, day 27 B1G ++++ B1W +++ B2R ++ B2G +++ B2W++++ Group C: C1R − VSP SNALP, day 27 C1G − C1B − C1W + C2R + C2G + C2W− Score: “−” = no visible tumor; “+” = evidence of tumor tissue atinjection site; “++” = Discrete tumor nodule protruding from liver lobe;“+++” = large tumor protruding on both sides of liver lobe; “++++” =large tumor, multiple nodules throughout liver lobe.

The correlation between body weights and tumor burden are shown in FIGS.4, 5 and 6.

A single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in theformation of mitotic spindles in liver tissue samples examined byhistological staining.

Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman).Human GAPDH was normalized to mouse GAPDH via species-specific Taqmanassays. Tumor score as shown by macroscopic observation in the tableabove correlated with GADPH levels (FIG. 7A).

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted bythe tumor. As described below, if levels of AFP go down after treatment,the tumor is not growing. Treatment with VSP lowered AFP levels in someanimals compared to treatment with controls (FIG. 7B).

Human HepB3 Study C:

In a third study, human HCC cells (HepB3) were injected directly intothe liver of SCID/beige mice, and treatment was initiated 20 days later.Group A animals were administered PBS; Group B animals were administered4 mg/kg Luc-1955 SNALP; Group C animals were administered 4 mg/kgSNALP-VSP; Group D animals were administered 2 mg/kg SNALP-VSP; andGroup E animals were administered 1 mg/kg SNALP-VSP. Treatment was witha single intravenous (iv) dose, and mice were sacrificed 24 hr. later.

Tumor burden and target silencing was assayed by qRT-PCR (Taqman). Tumorscore was also measured visually as described above, and the results areshown in the following table. hGAPDH levels, as shown in FIG. 8,correlates with macroscopic tumor score as shown in the table below.

TABLE 12 Tumor Burden by macroscopic Mouse observation Group A: PBS A2+++ A3 +++ A4 +++ Group B: 4 mg/kg Luc- B1 + 1955 SNALP B2 +++ B3 +++ B4+++ Group C: 4 mg/kg C1 ++ SNALP-VSP C2 ++ C3 ++ C4 +++ Group D: 2 mg/kgD1 ++ SNALP-VSP D2 + D3 + D4 ++ Group E: 1 mg/kg E1 +++ SNALP-VSP E2 +E3 ++ E4 + Score: “+” = variable tumor take/some small tumors; “++” =Discrete tumor nodule protruding from liver lobe; “+++” = large tumorprotruding on both sides of liver lobe

Human (tumor-derived) KSP silencing was assayed by Taqman analysis andthe results are shown in FIG. 10. hKSP expression was normalized tohGAPDH. About 80% tumor KSP silencing was observed at 4 mg/kg SNALP-VSP,and efficacy was evident at 1 mg/kg. The clear bars in FIG. 9 representthe results from small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was assayed by Taqman analysis andthe results are shown in FIG. 10. hVEGF expression was normalized tohGAPDH. About 60% tumor VEGF silencing was observed at 4 mg/kgSNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG.10 represent the results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was assayed by Taqman analysis andthe results are shown in FIG. 11A. mVEGF expression was normalized tohGAPDH. About 50% liver VEGF silencing was observed at 4 mg/kgSNALP-VSP, and efficacy was evident at 1 mg/kg.

Human HepB3 Study D: Contribution of Each dsRNA to Tumor Growth

In a fourth study, human HCC cells (HepB3) were injected directly intothe liver of SCID/beige mice, and treatment was initiated 8 days later.Treatment was with intravenous (iv) bolus injections, twice weekly, fora total of six does. The final dose was administered at day 25, and theterminal endpoint was at day 27.

Tumor burden was assayed by gross histology, human-specific mRNAanalysis (hGAPDH qPCR), and blood alpha-fetoprotein levels (serum AFPvia ELISA).

In Study 1, Group A was treated with PBS, Group B was treated withSNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).

In Study 2, Group A was treated with PBS; Group B was treated withSNALP-KSP+Luc (1 mg/kg), Group C was treated with ALN-VSP02 (1 mg/kg).

Both GAPDH mRNA levels and serum AFP levels were shown to decrease aftertreatment with ALN-VSP02 (FIG. 11B).

Histology Studies:

Human hepatoma Hep3B tumors were established by intrahepatic seeding inmice. SNALP treatment was initiated 20 days after tumor seeding.Tumor-bearing mice (three per group) were treated with a singleintravenous (IV) dose of (i) VSP SNALP or (ii) control (Luc) SNALP at 2mg/kg total siRNA.

Liver/tumor samples were collected for conventional H&E histology 24hours after single SNALP administration.

Large macroscopic tumor nodules (5-10 mm) were evident at necroscopy.

Effect of ALN-VSP in Hep3B Mice:

ALN-VSP (a cocktail of KSP dsRNA and VEGF dsRNA) treatment reduced tumorburden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels,a measure of tumor burden, were also observed to decline followingadministration of ALN-VSP dsRNA (see FIGS. 12A-12C). A decrease in tumorburden by visual macroscopic observation was also evident followingadministration of ALN-VSP.

A single IV bolus injection of ALN-VSP also resulted in mitotic spindleformation that was clearly detected in liver tissue samples from Hep3Bmice. This observation indicated cell cycle arrest.

Example 7 Survival of SNALP-VSP Animals Versus SNALP-Luc Treated Animals

To test the effect of siRNA SNALP on survival rates of cancer subjects,tumors were established by intrahepatic seeding in mice and the micewere treated with SNALP-siRNA. These studies utilized a VSP siRNAcocktail containing dsRNAs targeting KSP/Eg5 and VEGF. Control was dsRNAtargeting Luc. The siRNA cocktail was formulated in SNALPs.

Tumor cells (Human Hepatoma Hep3B, 1×10̂6) were injected directly intothe left lateral lobe of scid/beige mice. These mice were deficient forlymphocytes and natural killer (NK) cells, which is the minimal scopefor immune-mediated anti-tumor effects. The incisions were closed bysutures, and the mice allowed to recover for 2-5 hours. The mice werefully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose, and eachcocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1%DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug usingoriginal citrate buffer conditions.

siRNA-SNALP treatment was initiated on the day indicated below (18 or 26days) after tumor seeding. siRNA-SNALP were administered twice a weekfor three weeks after 18 or 26 days at a dose of 4 mg/kg. Survival wasmonitored and animals were euthanized based on humane surrogateendpoints (e.g., animal body weight, abdominal distension/discoloration,and overall health).

The survival data for treatment initiated 18 days after tumor seeing issummarized in Table 13, Table 14, and FIG. 13A.

TABLE 13 Kaplan-Meier (survival) data (% Surviving) SNALP- SNALP- DayLuc VSP 18 100% 100% 22 100% 100% 25 100% 100% 27 100% 100% 28 100% 100%28 86% 100% 29 86% 100% 32 86% 100% 33 86% 100% 33 43% 100% 35 43% 100%36 43% 100% 36 29% 100% 38 29% 100% 38 14% 100% 38 14% 88% 40 14% 88% 4314% 88% 45 14% 88% 49 14% 88% 51 14% 88% 51 14% 50% 53 14% 50% 53 14%25% 55 14% 25% 57 14% 25% 57 0% 0%

TABLE 14 Survival in days, for each animal. Treatment Animal groupSurvival 1 SNALP-Luc 28 days 2 SNALP-Luc 33 days 3 SNALP-Luc 33 days 4SNALP-Luc 33 days 5 SNALP-Luc 36 days 6 SNALP-Luc 38 days 7 SNALP-Luc 57days 8 SNALP-VSP 38 days 9 SNALP-VSP 51 days 10 SNALP-VSP 51 days 11SNALP-VSP 51 days 12 SNALP-VSP 53 days 13 SNALP-VSP 53 days 14 SNALP-VSP57 days 15 SNALP-VSP 57 days

FIG. 13A shows the mean survival of SNALP-VSP animals and SNALP-Luctreated animals versus days after tumor seeding. The mean survival ofSNALP-VSP animals was extended by approximately 15 days versus SNALP-Luctreated animals.

TABLE 15 Serum alpha fetoprotein (AFP) concentration, for each animal,at a time pre-treatment and at end of treatment (concentration in μg/ml)End of pre-Rx Rx 1 SNALP-Luc 30.858 454.454 2 SNALP-Luc 10.088 202.082 3SNALP-Luc 23.736 648.952 4 SNALP-Luc 1.696 13.308 5 SNALP-Luc 4.778338.688 6 SNALP-Luc 15.004 826.972 7 SNALP-Luc 11.036 245.01 8 SNALP-VSP37.514 182.35 9 SNALP-VSP 91.516 248.06 10 SNALP-VSP 25.448 243.13 11SNALP-VSP 24.862 45.514 12 SNALP-VSP 57.774 149.352 13 SNALP-VSP 12.44678.724 14 SNALP-VSP 2.912 9.61 15 SNALP-VSP 4.516 11.524

Tumor burden was monitored using serum AFP levels during the course ofthe experiment. Alpha-fetoprotein (AFP) is a major plasma proteinproduced by the yolk sac and the liver during fetal life. The protein isthought to be the fetal counterpart of serum albumin, and human AFP andalbumin gene are present in tandem in the same transcriptionalorientation on chromosome 4. AFP is found in monomeric as well asdimeric and trimeric forms, and binds copper, nickel, fatty acids andbilirubin. AFP levels decrease gradually after birth, reaching adultlevels by 8-12 months. Normal adult AFP levels are low, but detectable.AFP has no known function in normal adults and AFP expression in adultsis often associated with a subset of tumors such as hepatoma andteratoma. AFP is a tumor marker used to monitor testicular cancer,ovarian cancer, and malignant teratoma. Principle tumors that secreteAFP include endodermal sinus tumor (yolk sac carcinoma), neuroblastoma,hepatoblastoma, and heptocellular carcinoma. In patients withAFP-secreting tumors, serum levels of AFP often correlate with tumorsize. Serum levels are useful in assessing response to treatment.Typically, if levels of AFP go down after treatment, the tumor is notgrowing. A temporary increase in AFP immediately following chemotherapymay indicate not that the tumor is growing but rather that it isshrinking (and releasing AFP as the tumor cells die). Resection isusually associated with a fall in serum levels. As shown in FIG. 14,tumor burden in SNALP-VSP treated animals was significantly reduced.

The experiment was repeated with SNALP-siRNA treatment at 26, 29, 32 35,39, and 42 days after implantation. The data is shown in FIG. 13B. Themean survival of SNALP-VSP animals was extended by approximately 15 daysversus SNALP-Luc treated animals by approximately 19 days, or 38%.

Example 8 Induction of Mono-Asters in Established Tumors

Inhibition of KSP in dividing cells leads to the formation of monoasters that are readily observable in histological sections. Todetermine whether mono aster formation occurred in SNALP-VSP treatedtumors, tumor bearing animals (three weeks after Hep3B cellimplantation) were administered 2 mg/kg SNALP-VSP via tail veininjection. Control animals received 2 mg/kg SNALP-Luc. Twenty four hourslater, animals were sacrificed, and tumor bearing liver lobes wereprocessed for histological analysis. Representative images of H&Estained tissue sections are shown in FIG. 15. Extensive mono asterformation was evident in ALN VSP02 treated (A), but not SNALP-Luctreated (B), tumors. In the latter, normal mitotic figures were evident.The generation of mono asters is a characteristic feature of KSPinhibition and provides further evidence that SNALP-VSP has significantactivity in established liver tumors.

Example 9 Manufacturing Process and Product Specification of ALN-VSP02(SNALP-VSP)

ALN-VSP02 product contains 2 mg/mL of drug substance ALN-VSPDS01formulated in a sterile lipid particle formulation (referred to asSNALP) for IV administration via infusion. Drug substance ALN-VSPDS01consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targetingVEGF) in an equimolar ratio. The drug product is packaged in 10 mL glassvials with a fill volume of 5 mL.

The following terminology is used herein:

Drug Substance siRNA Duplexes Single Strand Intermediates ALN-VSPDS01ALN-12115* Sense: A-19562 Antisense: A-19563 ALN-3133** Sense: A-3981Antisense: A-3982 *Alternate names = AD-12115, AD12115; **Alternatenames = AD-3133, AD3133

9.1 Preparation of Drug Substance ALN-VSPDS01

The two siRNA components of drug substance ALN-VSPDS01, ALN-12115 andALN-3133, are chemically synthesized using commercially availablesynthesizers and raw materials. The manufacturing process consists ofsynthesizing the two single strand oligonucleotides of each duplex (A19562 sense and A 19563 antisense of ALN 12115 and A 3981 sense and A3982 antisense of ALN 3133) by conventional solid phase oligonucleotidesynthesis using phosphoramidite chemistry and 5′ Odimethoxytriphenylmethyl (DMT) protecting group with the 2′ hydroxylprotected with tert butyldimethylsilyl (TBDMS) or the 2′ hydroxylreplaced with a 2′ methoxy group (2′ OMe). Assembly of anoligonucleotide chain by the phosphoramidite method on a solid supportsuch as controlled pore glass or polystyrene. The cycle consists of 5′deprotection, coupling, oxidation, and capping. Each coupling reactionis carried out by activation of the appropriately protected ribo, 2′OMe, or deoxyribonucleoside amidite using 5 (ethylthio) 1H tetrazolereagent followed by the coupling of the free 5′ hydroxyl group of asupport immobilized protected nucleoside or oligonucleotide. After theappropriate number of cycles, the final 5′ protecting group is removedby acid treatment. The crude oligonucleotide is cleaved from the solidsupport by aqueous methylamine treatment with concomitant removal of thecyanoethyl protecting group as well as nucleobase protecting groups. The2′ O TBDMS group is then cleaved using a hydrogen fluoride containingreagent to yield the crude oligoribonucleotide, which is purified usingstrong anion exchange high performance liquid chromatography (HPLC)followed by desalting using ultrafiltration. The purified single strandsare analyzed to confirm the correct molecular weight, the molecularsequence, impurity profile and oligonucleotide content, prior toannealing into the duplexes. The annealed duplex intermediates ALN 12115and ALN 3133 are either lyophilized and stored at 20° C. or mixed in 1:1molar ratio and the solution is lyophilized to yield drug substance ALNVSPDS01. If the duplex intermediates were stored as dry powder, they areredissolved in water before mixing. The equimolar ratio is achieved bymonitoring the mixing process by an HPLC method.

The manufacturing process flow diagram is shown in FIG. 16.

Example specifications are shown in Table 16a.

The results of up to 12 month stability testing for ALN-VSPDS01 drugsubstance are shown in Tables 16c. The assay methods were chosen toassess physical property (appearance, pH, moisture), purity (by SEC anddenaturing anion exchange chromatography) and potency (by denaturinganion exchange chromatography [AX-HPLC]).

TABLE 16a Example specifications for ALN-VSPDS01 Test Method AcceptanceCriteria Appearance Visual White to off-white powder Identity,ALN-VSPDS01 Duplex AX-HPLC Duplex retention times are consistentALN-3133 with those of reference standards ALN-12115 Identity,ALN-VSPDS01 MS Molecular weight of single strands are within thefollowing ranges: A-3981: 6869-6873 Da A-3982: 7305-7309 Da A-19562:6762-6766 Da A-19563: 6675-6679 Da Sodium counter ion (% w/w on FlameAAS or ICP-OES Report data anhydrous basis) ALN-VSPDS01 assay DenaturingAX-HPLC 90-110% Purity of ALN-VSPDS01 SEC ≧90.0 area % Single strandpurity, Denaturing AX-HPLC Report data ALN-VSPDS01 Report area % fortotal impurities siRNA molar ratio Duplex AX-HPLC 1.0 ± 0.1 Moisturecontent Karl Fischer titration ≦15% Residual solvents HS-Capillary GCAcetonitrile ≦410 ppm Ethanol ≦5000 ppm Isopropanol ≦5000 ppm pH of 1%solution USP <791> Report data Heavy metals ICP-MS Report data As, Cd,Cu, Cr, Fe, Ni, Pb, Sn Bacterial endotoxins USP <85> ≦0.5 EU/mgBioburden Modified USP <61> <100 CFU/g

TABLE 16b Stability of drug substance Lot No.: A05M07001N Study StorageConditions: −20° C. (Storage Condition) Acceptance Results Test MethodCriteria Initial 1 Month 3 Months 6 Months 12 Months Appearance VisualWhite to off- Pass Pass Pass Pass Pass white powder pH USP <791> Reportdata 6.7 6.4 6.6 6.4 6.8 Moisture Karl Fischer ≦15% 3.6* 6.7 6.2 5.6 5.0content titration (% w/w) Purity (area SEC ≧90.0 area % 95 95 94 92 95%) A-3981 Denaturing AX- Report data 24 23 23 23 23 (sense) HPLC (area%) A-3982 Denaturing AX- Report data 23 23 23 23 24 (antisense) HPLC(area %) A-19562 Denaturing AX- Report data 22 21 21 21 21 (sense) HPLC(area %) A-19563 Denaturing AX- Report data 23 22 22 22 22 (antisense)HPLC (area %)

9.2 Preparation of Drug Product ALN-VSP02 (SNALP-VSP)

ALN VSP02, is a sterile formulation of the two siRNAs (in a 1:1 molarratio) with lipid excipients in isotonic buffer. The lipid excipientsassociate with the two siRNAs, protect them from degradation in thecirculatory system, and aid in their delivery to the target tissue. Thespecific lipid excipients and the quantitative proportion of each (shownin Table 17) have been selected through an iterative series ofexperiments comparing the physicochemical properties, stability,pharmacodynamics, pharmacokinetics, toxicity and productmanufacturability of numerous different formulations. The excipientDLinDMA is a titratable aminolipid that is positively charged at low pH,such as that found in the endosome of mammalian cells, but relativelyuncharged at the more neutral pH of whole blood. This featurefacilitates the efficient encapsulation of the negatively charged siRNAsat low pH, preventing formation of empty particles, yet allows foradjustment (reduction) of the particle charge by replacing theformulation buffer with a more neutral storage buffer prior to use.Cholesterol and the neutral lipid DPPC are incorporated in order toprovide physicochemical stability to the particles. Thepolyethyleneglycol lipid conjugate PEG2000 C DMA aids drug productstability, and provides optimum circulation time for the proposed use.ALN VSP02 lipid particles have a mean diameter of approximately 80-90 nmwith low polydispersity values. A representative cryo transmissionelectron microscope (cryo TEM) image is shown in FIG. 17. At neutral pH,the particles are essentially uncharged, with Zeta Potential values ofless than 6 mV. There is no evidence of empty (non loaded) particlesbased on the manufacturing process.

TABLE 17 Quantitative Composition of ALN-VSP02 Component, gradeProportion (mg/mL) ALN-VSPDS01, cGMP 2.0* DLinDMA 7.3(1,2-Dilinoleyloxy- N,N-dimethyl-3-aminopropane), cGMP DPPC(R-1,2-Dipalmitoyl-sn-glycero-3- 1.1 phosphocholine), cGMP Cholesterol,Synthetic, cGMP 2.8 PEG2000-C-DMA 0.8 (3-N-[(ω-Methoxy poly(ethyleneglycol) 2000)carbamoyl]-1,2-dimyristyloxy- propylamine), cGMP PhosphateBuffered Saline, cGMP q.s. *The 1:1 molar ratio of the two siRNAs in thedrug product is maintained throughout the size distribution of the drugproduct particles.

Solutions of lipid (in ethanol) and ALN VSPDS01 drug substance (inaqueous buffer) are mixed and diluted to form a colloidal dispersion ofsiRNA lipid particles with an average particle size of approximately80-90 nm. This dispersion is then filtered through 0.45/0.2 μm filters,concentrated, and diafiltered by Tangential Flow Filtration. After inprocess testing and concentration adjustment to 2.0 mg/mL, the productis sterile filtered, aseptically filled into glass vials, stoppered,capped and placed at 5±3° C. The ethanol and all aqueous buffercomponents are USP grade; all water used is USP Sterile Water ForInjection grade. Representative ALN-VSP02 process is shown in flowdiagram in FIG. 18.

TABLE 18a Example ALN-VSP02 specifications Test Analytical ProcedureAcceptance Criteria Appearance Visual White to off-white, homogeneousopalescent liquid, no foreign particles pH USP <791> 6.8-7.8 OsmolalityUSP <785> 250-350 mOsm/kg Identity, ALN-VSPDS01 Duplex Retention timesconsistent with ALN-3133 Anion Exchange (AX)-HPLC those of referencestandards ALN-12115 Identity, ALN-VSPDS01 Denaturing Retention timesconsistent with A-3981 AX-HPLC those of reference standards A-3982A-19562 A-19563 Lipid identity Reversed Phase (RP)-HPLC with Retentiontimes consistent with DLinDMA Evaporative Light Scattering those ofreference standards PEG₂₀₀₀-C-DMA (ELS) detection DPPC CholesterolALN-VSPDS01 label claim Denaturing 1.7-2.3 mg/mL (Strength/Potency)AX-HPLC Duplex molar ratio Duplex 1.0 ± 0.1 AX-HPLC DLinDMA contentRP-HPLC with 5.6-10.3 mg/mL ELS detection PEG₂₀₀₀-C-DMA content RP-HPLCwith 0.6-1.1 mg/mL ELS detection DPPC content RP-HPLC with 0.8-1.5 mg/mLELS detection Cholesterol content RP-HPLC with 2.1-3.9 mg/mL ELSdetection Total lipid:ALN-VSPDS01 ratio Calculated from total lipidassay 4.9-8.1 mg/mg and label claim for drug substance ALN-VSPDS01encapsulation Fluorometric assay ≧90.0% Purity Denaturing ≧80.0 area %AX-HPLC Impurity profile Denaturing Report retention times (relative toAX-HPLC A-19563) and area % for all peaks ≧0.20% Residual ethanol USP<467> ≦5000 ppm Residual EDTA Ion Pairing (IP)-HPLC with UV ≦2000 μg/mLdetection Particle size Z average Dynamic light scattering 60-120 nmPolydispersity Dynamic light scattering ≦0.15 Particle size distributionDynamic light scattering Report data D₁₀ D₅₀ D₉₀ Particulate matterModified USP <788> ≧25 μm ≦300 per container ≧10 μm ≦3000 per containerBacterial endotoxins Modified USP <85> ≦5.0 EU/mL Sterility USP <71>Pass Volume in container USP <1> ≧5.0 mL Dose uniformity USP <905> PassHeavy metal analysis Inductive Coupled Plasma Mass Report dataSpectrometry (ICP-MS)

9.4 Container/Closure System

The ALN VSP02 drug product is packaged in 10 mL glass vials with a fillvolume of 5 mL. The container closure system is comprised of a USP/EPType I borosilicate glass vial, a teflon faced butyl rubber stopper andan aluminum flip off cap. The drug product will be stored at 5±3° C.

9.5 Stability of Drug Product ALN-VSP02

Stability data (25° C./60% RH) are given in Table 18b and 18c.

TABLE 18b Example ALN-VSP02 stability at storage conditions StudyStorage Conditions: 2-8° C. Lot No.: IC097 Results Acceptance 1 2 3 4 6Test Method Criteria Initial Month Months Months Months MonthsAppearance Visual White to off-white, Pass Pass Pass Pass Pass Passhomogeneous opalescent liquid, no foreign particles pH USP 6.8-7.8 7.47.4 7.4 7.3 7.4 7.3 <791> Osmolality USP 250-350 mOsm/kg 308 307 305 306309 305 <785> ALN- Duplex Retention times Pass Pass Pass Pass Pass PassVSPDS01 AX-HPLC consistent with Identity, those of reference ALN-3133standards ALN- 12115 ALN- Denaturing Retention times Pass Pass Pass PassPass Pass VSPDS01 AX-HPLC consistent with Identity, those of referenceA-3981 standards A-3982 A-19562 A-19563 Lipid RP-HPLC Retention timesPass Pass Pass Pass Pass Pass identity, with ELS consistent with DLinDMADetection those of reference PEG₂₀₀₀- standards C-DMA DPPC CholesterolALN- Denaturing 1.7-2.3 mg/mL 2.1 2.2 2.1 2.1 2.1 2.1 VSPDS01 AX-HPLCstrength/potency Duplex Duplex 1.0 ± 0.1 1.0 1.0 1.0 1.0 1.0 1.0 molarratio AX-HPLC DLinDMA RP-HPLC 5.6-10.3 mg/mL 9.1 9.4 9.1 9.6 9.1 9.2content with ELS Detection Cholesterol RP-HPLC 2.1-3.9 mg/mL 3.4 3.5 3.43.5 3.4 3.5 content with ELS Detection DPPC RP-HPLC 0.8-1.5 mg/mL 1.31.3 1.4 1.4 1.2 1.3 content with ELS Detection PEG₂₀₀₀- RP-HPLC 0.6-1.1mg/mL 1.0 1.0 1.0 1.1 1.0 1.0 C-DMA with ELS content Detection TotalCalculation 4.9-8.1 mg/mg 7.0 6.9 7.1 7.4 7.0 7.1 lipid:ALN- VSPDS01ratio ALN- Fluorometric ≧90.0% 95.9 96.5 94.4 98.1 97.8 96.4 VSPDS01assay encapsulation Purity Denaturing ≧80.0% 90.7 89.6 90.8 91.3 92.490.8 AX-HPLC Particle Light 60-120 nm 86 87 87 87 87 87 size, scatteringZ-average Polydispersity Light ≦0.15 0.02 0.03 0.02 0.03 0.03 0.03scattering Particle Light Report data (nm) 56 56 56 56 56 56 sizescattering distribution, D₁₀ Particle Light Report data (nm) 76 77 77 7778 77 size scattering distribution, D₅₀ Particle Light Report data (nm)110 112 112 113 112 113 size scattering distribution, D₉₀ ParticulateModified (per container) matter, USP ≧25 μm <788> ≦300 18 NS NS NS NS 3≧10 μm ≦3000 48 11 Bacterial USP <85> ≦5.0 EU/mL 0.50 NS NS NS NS NSendotoxins Sterility USP <71> Pass Pass NS NS NS NS NS

TABLE 18c Example ALN-VSP02 stability at 25° C./ambient humidity StudyStorage Conditions: 25° C./ambient humidity Lot No.: IC097 ResultsAcceptance 1 2 3 4 6 Test Method Criteria Initial Month Months MonthsMonths Months Appearance Visual White to off-white, Pass Pass Pass PassPass Pass homogeneous opalescent liquid, no foreign particles pH USP<791> 6.8-7.8 7.4 7.3 7.2 7.1 7.2 7.1 Osmolality USP <785> 250-350mOsm/kg 308 306 304 307 307 304 ALN- Duplex Retention times Pass PassPass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity, those ofreference ALN-3133 standards ALN- 12115 ALN- Denaturing Retention timesPass Pass Pass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity,those of reference A-3981 standards A-3982 A-19562 A-19563 Lipid RP-HPLCRetention times Pass Pass Pass Pass Pass Pass identity, with ELSconsistent with DLinDMA Detection those of reference PEG₂₀₀₀- standardsC-DMA DPPC Cholesterol ALN- Denaturing 1.7-2.3 mg/mL 2.1 2.1 2.0 2.0 2.02.0 VSPDS01 AX-HPLC strength/potency Duplex Duplex 1.0 ± 0.1 1.0 1.0 1.01.0 1.0 1.0 molar ratio AX-HPLC DLinDMA RP-HPLC 5.6-10.3 mg/mL 9.1 9.69.0 9.3 9.2 9.3 content with ELS Detection Cholesterol RP-HPLC 2.1-3.9mg/mL 3.4 3.5 3.4 3.5 3.4 3.5 content with ELS Detection DPPC RP-HPLC0.8-1.5 mg/mL 1.3 1.3 1.3 1.2 1.2 1.1 content with ELS DetectionPEG₂₀₀₀- RP-HPLC 0.6-1.1 mg/mL 1.0 1.0 1.0 1.1 1.0 1.0 C-DMA with ELScontent Detection Total Calculation 4.9-8.1 mg/mg 7.0 7.3 7.4 7.6 7.47.5 lipid:ALN- VSPDS01 ratio ALN- Fluorometric ≧90.0% 95.9 97.2 94.697.9 97.9 96.7 VSPDS01 assay encapsulation Purity Denaturing ≧80.0% 90.788.0 88.9 88.4 89.0 85.3 AX-HPLC Particle Light 60-120 nm 86 85 86 89 8787 size, scattering Z-average Polydispersity Light ≦0.15 0.02 0.05 0.030.04 0.04 0.03 scattering Particle Light Report data (nm) 56 54 56 58 5657 size scattering distribution, D₁₀ Particle Light Report data (nm) 7675 77 79 77 78 size scattering distribution, D₅₀ Particle Light Reportdata (nm) 110 110 111 116 113 113 size scattering distribution, D₉₀Particulate Modified (per container) matter, USP <788> ≧25 μm ≦300 18 NSNS NS NS 1 ≧10 μm ≦3000 48 16 Bacterial USP <85> ≦5.0 EU/mL 0.50 NS NSNS NS <0.50 endotoxins Sterility USP <71> Pass Pass NS NS NS NS Pass

Example 10 In Vitro Efficacy of ALN-VSP02 in Human Cancer Cell Lines

The efficacy of ALN-VSP02 treatment in human cancer cell lines wasdetermined via measurement of KSP mRNA, VEGF mRNA, and cell viabilityafter treatment. IC50 (nM) values determined for KSP and VEGF in eachcell line.

TABLE 19 cell lines Cell line tested ATCC cat number HELA ATCC Cat N:CCL-2 KB ATCC Cat N: CCL-17 HEP3B ATCC Cat N: HB-8064 SKOV-3 ATCC Cat N:HTB-77 HCT-116 ATCC Cat N: CCL-247 HT-29 ATCC Cat N: HTB-38 PC-3 ATCCCat N: CRL-1435 A549 ATCC Cat N: CCL-185 MDA-MB-231 ATCC Cat N: HTB-26

Cells were plated in 96 well plates in complete media at day 1 to reacha density of 70% on day 2. On day 2 media was replaced with Opti-MEMreduced serum media (Invitrogen Cat N: 11058-021) and cells weretransfected with either ALN-VSP02 or control SNALP-Luc withconcentration range starting at 1.8 μM down to 10 pM. After 6 hours themedia was changed to complete media. Three replicate plates for eachcell line for each experiment was done.

Cells were harvested 24 hours after transfection. KSP levels weremeasured using bDNA; VEGF mRNA levels were measured using human TaqManassay.

Viability was measured using Cell Titer Blue reagent (Promega Cat N:G8080) at 48 and/or 72 h following manufacturer's recommendations.

As shown in Table 20, nM concentrations of VSP02 are effective inreducing expression of both KSP and VEGF in multiple human cell lines.Viability of treated cells was not

TABLE 20 Results IC50 (nM) IC50 (nM) Cell line KSP VEGF HeLa 8.79 672SKOV-3 142 1347 HCT116 31.6 27.5 Hep3B 1.3 14.5 HT-29 262 ND PC3 127 NDKB 50.6 ND A549 201 ND MB231 187 ND

Example 11 Anti-Tumor Efficacy of VSP SNALP vs. Sorafenib in EstablishedHep3B Intrahepatic Tumors

The anti-tumor effects of multi-dosing VSP SNALP verses Sorafenib inscid/beige mice bearing established Hep3B intrahepatic tumors wasstudied. Sorafenib is a small molecule inhibitor of protein kinasesapproved for treatment of hepatic cellular carcinoma (HCC).

Tumors were established by intrahepatic seeding in scid/beige mice asdescribed herein. Treatment was initiated 11 days post-seeding. Micewere treated with Sorafenib and a control siRNA-SNALP, Sorafenib and VSPsiRNA-SNALP, or VSP siRNA-SNALP only. Control mice were treated withbuffers only (DMSO for Sorafenib and PBS for siRNA-SNALP). Sorafenib wasadministered intraparenterally from Mon to Fri for three weeks, at 15mg/kg according to body weight for a total of 15 injections. Sorafenibwas administered a minimum of 1 hour after SNALP injections. ThesiRNA-SNALPS were administered intravenously via the lateral tail veinaccording at 3 mg/kg based on the most recently recorded body weight (10ml/kg) for 3 weeks (total of 6 doses) on days 1, 4, 7, 10, 14, and 17.

Mice were euthanized based on an assessment of tumor burden includingprogressive weight loss and clinical signs including condition,abdominal distension/discoloration and mobility.

The percent survival data are shown in FIG. 19. Co-administration of VSPsiRNA-SNALP with Sorafenib increased survival proportion compared toadministration of Sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALPincreased survival proportion compared to Sorafenib.

Example 12 In Vitro Efficacy of VSP Using Variants of AD-12115 andAD-3133

Two sets of duplexes targeted to Eg5/KSP and VEGF were designed andsynthesized. Each set included duplexes tiling 10 nucleotides in eachdirection of the target sites for either AD-12115 and AD-3133.

Sequences of the target, sense strand, and antisense strand for eachduplex are shown in the Table below.

Each duplex is assayed for inhibition of expression using the assaysdescribed herein. The duplexes are administered alone and/or incombination, e.g., an Eg5/KSP dsRNA in combination with a VEGF dsRNA. Insome embodiments, the dsRNA are administered in a SNALP formulation asdescribed herein.

TABLE 21 Sequences of dsRNA targeted to VEGF and Eg5/KSP (tiling) SEQSense Strand SEQ target target sequence ID Antisense strand ID Duplex IDgene 5′ to 3′ NO: 5′ to 3′ NO: AD- VEGFA ACCAAGGCCAGCACAUAGG 2264AccAAGGccAGcAcAuAGGTsT 2304 20447.1 CCuAUGUGCUGGCCUUGGUTsT 2305 AD-VEGFA CCAAGGCCAGCACAUAGGA 2265 ccAAGGccAGcAcAuAGGATsT 2306 20448.1UCCuAUGUGCUGGCCUUGGTsT 2307 AD- VEGFA CCAAGGCCAGCACAUAGGA 2266ccAAGGccAGcAcAuAGGATsT 2308 20449.1 CUCCuAUGUGCUGGCCUUGTsT 2309 AD-VEGFA AAGGCCAGCACAUAGGAGA 2267 AAGGccAGcAcAuAGGAGATsT 2310 20450.1UCUCCuAUGUGCUGGCCUUTsT 2311 AD- VEGFA AGGCCAGCACAUAGGAGAG 2268AGGccAGcAcAuAGGAGAGTsT 2312 20451.1 CUCUCCuAUGUGCUGGCCUTsT 2313 AD-VEGFA GGCCAGCACAUAGGAGAGA 2269 GGccAGcAcAuAGGAGAGATsT 2314 20452.1UCUCUCCuAUGUGCUGGCCTsT 2315 AD- VEGFA GCCAGCACAUAGGAGAGAU 2270GccAGcAcAuAGGAGAGAuTsT 2316 20453.1 2317 AD- VEGFA CCAGCACAUAGGAGAGAUG2271 ccAGcAcAuAGGAGAGAuGTsT 2318 20454.1 cAUCUCUCCuAUGUGCUGGTsT 2319 AD-VEGFA CAGCACAUAGGAGAGAUGA 2272 cAGcAcAuAGGAGAGAuGATsT 2320 20455.1UcAUCUCUCCuAUGUGCUGTsT 2321 AD- VEGFA AGCACAUAGGAGAGAUGAG 2273AGcAcAuAGGAGAGAuGAGTsT 2322 20456.1 CUcAUCUCUCCuAUGUGCUTsT 2323 AD-VEGFA CACAUAGGAGAGAUGAGCU 2274 cAcAuAGGAGAGAuGAGcuTsT 2324 20457.1AGCUcAUCUCUCCuAUGUGTsT 2325 AD- VEGFA ACAUAGGAGAGAUGAGCUU 2275AcAuAGGAGAGAuGAGcuuTsT 2326 20458.1 AAGCUcAUCUCUCCuAUGUTsT 2327 AD-VEGFA CAUAGGAGAGAUGAGCUUC 2276 cAuAGGAGAGAuGAGcuucTsT 2328 20459.1GAAGCUcAUCUCUCCuAUGTsT 2329 AD- VEGFA AUAGGAGAGAUGAGCUUCC 2277AuAGGAGAGAuGAGcuuccTsT 2330 20460.1 GGAAGCUcAUCUCUCCuAUTsT 2331 AD-VEGFA UAGGAGAGAUGAGCUUCCU 2278 uAGGAGAGAuGAGcuuccuTsT 2332 20461.1AGGAAGCUcAUCUCUCCuATsT 2333 AD- VEGFA AGGAGAGAUGAGCUUCCUA 2279AGGAGAGAuGAGcuuccuATsT 2334 20462.1 uAGGAAGCUcAUCUCUCCUTsT 2335 AD-VEGFA GGAGAGAUGAGCUUCCUAC 2280 GGAGAGAuGAGcuuccuAcTsT 2336 20463.1GuAGGAAGCUcAUCUCUCCTsT 2337 AD- VEGFA GAGAGAUGAGCUUCCUACA 2281GAGAGAuGAGcuuccuAcATsT 2338 20464.1 UGuAGGAAGCUcAUCUCUCTsT 2339 AD-VEGFA AGAGAUGAGCUUCCUACAG 2282 AGAGAuGAGcuuccuAcAGTsT 2340 20465.1CUGuAGGAAGCUcAUCUCUTsT 2341 AD- VEGFA GAGAUGAGCUUCCUACAGC 2283GAGAuGAGcuuccuAcAGcTsT 2342 20466.1 GCUGuAGGAAGCUcAUCUCTsT 2343 AD- KSPAUGUUCCUUAUCGAGAAUC 2284 AuGuuccuuAucGAGAAucTsT 2344 20467.1GAUUCUCGAuAAGGAAcAUTsT 2345 AD- KSP UGUUCCUUAUCGAGAAUCU 2285uGuuccuuAucGAGAAucuTsT 2346 20468.1 2347 AD- KSP GUUCCUUAUCGAGAAUCUA2286 GuuccuuAucGAGAAucuATsT 2348 20469.1 uAGAUUCUCGAuAAGGAACTsT 2349 AD-KSP UUCCUUAUCGAGAAUCUAA 2287 uuccuuAucGAGAAucuAATsT 2350 20470.1UuAGAUUCUCGAuAAGGAATsT 2351 AD- KSP UCCUUAUCGAGAAUCUAAA 2288uccuuAucGAGAAucuAAATsT 2352 20471.1 UUuAGAUUCUCGAuAAGGATsT 2353 AD- KSPCCUUAUCGAGAAUCUAAAC 2289 ccuuAucGAGAAucuAAAcTsT 2354 20472.1GUUuAGAUUCUCGAuAAGGTsT 2355 AD- KSP CUUAUCGAGAAUCUAAACU 2290cuuAucGAGAAucuAAAcuTsT 2356 20473.1 AGUUuAGAUUCUCGAuAAGTsT 2357 AD- KSPUUAUCGAGAAUCUAAACUA 2291 uuAucGAGAAucuAAAcuATsT 2358 20474.1uAGUUuAGAUUCUCGAuAATsT 2359 AD- KSP UAUCGAGAAUCUAAACUAA 2292uAucGAGAAucuAAAcuAATsT 2360 20475.1 UuAGUUuAGAUUCUCGAuATsT 2361 AD- KSPAUCGAGAAUCUAAACUAAC 2293 AucGAGAAucuAAAcuAAcTsT 2362 20476.1GUuAGUUuAGAUUCUCGAUTsT 2363 AD- KSP CGAGAAUCUAAACUAACUA 2294cGAGAAucuAAAcuAAcuATsT 2364 20477.1 uAGUuAGUUuAGAUUCUCGTsT 2365 AD- KSPGAGAAUCUAAACUAACUAG 2295 GAGAAucuAAAcuAAcuAGTsT 2366 20478.1CuAGUuAGUUuAGAUUCUCTsT 2367 AD- KSP AGAAUCUAAACUAACUAGA 2296AGAAucuAAAcuAAcuAGATsT 2368 20479.1 UCuAGUuAGUUuAGAUUCUTsT 2369 AD- KSPGAAUCUAAACUAACUAGAA 2297 GAAucuAAAcuAAcuAGAATsT 2370 20480.1UUCuAGUuAGUUuAGAUUCTsT 2371 AD- KSP AAUCUAAACUAACUAGAAU 2298AAucuAAAcuAAcuAGAAuTsT 2372 20481.1 AUUCuAGUuAGUUuAGAUUTsT 2373 AD- KSPAUCUAAACUAACUAGAAUC 2299 AucuAAAcuAAcuAGAAucTsT 2374 20482.1GAUUCuAGUuAGUUuAGAUTsT 2375 AD- KSP UCUAAACUAACUAGAAUCC 2300ucuAAAcuAAcuAGAAuccTsT 2376 20483.1 2377 AD- KSP CUAAACUAACUAGAAUCCU2301 cuAAAcuAAcuAGAAuccuTsT 2378 20484.1 AGGAUUCuAGUuAGUUuAGTsT 2379 AD-KSP UAAACUAACUAGAAUCCUC 2302 uAAAcuAAcuAGAAuccucTsT 2380 20485.1GAGGAUUCuAGUuAGUUuATsT 2381 AD- KSP AAACUAACUAGAAUCCUCC 2303AAAcuAAcuAGAAuccuccTsT 2382 20486.1 GGAGGAUUCuAGUuAGUUUTsT 2383

Example 13 VEGF Targeted dsRNA with a Single Blunt End

A set duplexes targeted to VEGF were designed and synthesized. The setincluded duplexes tiling 10 nucleotides in each direction of the targetsites for AD-3133. Each duplex includes a 2 base overhang at the endcorresponding to the 3′ end of the antisense strand and no overhang,e.g., a blunt end, at the end corresponding to the 5′ end of theantisense strand.

The sequences of each strand of these duplexes are shown in thefollowing table.

Each duplex is assayed for inhibition of expression using the assaysdescribed herein. The VEGF duplexes are administered alone and/or incombination with an Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments,the dsRNA are administered in a SNALP formulation as described herein.

TABLE 22 Target sequences of blunt ended dsRNA targeted to VEGF SEQ IDVEGF target sequence position on duplex ID NO: 5′ to 3′ VEGF geneAD-20447.1 2384 ACCAAGGCCAGCACAUAGG 1365 AD-20448.1 2385CCAAGGCCAGCACAUAGGA 1366 AD-20449.1 2386 CAAGGCCAGCACAUAGGAG 1367AD-20450.1 2387 AAGGCCAGCACAUAGGAGA 1368 AD-20451.1 2388AGGCCAGCACAUAGGAGAG 1369 AD-20452.1 2389 GGCCAGCACAUAGGAGAGA 1370AD-20453.1 2390 GCCAGCACAUAGGAGAGAU 1371 AD-20454.1 2391CCAGCACAUAGGAGAGAUG 1372 AD-20455.1 2392 CAGCACAUAGGAGAGAUGA 1373AD-20456.1 2393 AGCACAUAGGAGAGAUGAG 1374 AD-20457.1 2394CACAUAGGAGAGAUGAGCU 1376 AD-20458.1 2395 ACAUAGGAGAGAUGAGCUU 1377AD-20459.1 2396 CAUAGGAGAGAUGAGCUUC 1378 AD-20460.1 2397AUAGGAGAGAUGAGCUUCC 1379 AD-20461.1 2398 UAGGAGAGAUGAGCUUCCU 1380AD-20462.1 2399 AGGAGAGAUGAGCUUCCUA 1381 AD-20463.1 2400GGAGAGAUGAGCUUCCUAC 1382 AD-20464.1 2401 GAGAGAUGAGCUUCCUACA 1383AD-20465.1 2402 AGAGAUGAGCUUCCUACAG 1384 AD-20466.1 2403GAGAUGAGCUUCCUACAGC 1385

TABLE 23 Strand sequences of blunt ended dsRNA targeted to VEGF SEQ SEQSense strand ID Antisense strand ID duplex ID (5′ to 3′) NO: (5′ to 3′)NO: AD-20447.1 ACCAAGGCCAGCACAUAGGAG 2404 CUCCUAUGUGCUGGCCUUGGUGA 2424AD-20448.1 CCAAGGCCAGCACAUAGGAGA 2405 UCUCCUAUGUGCUGGCCUUGGUG 2425AD-20449.1 CAAGGCCAGCACAUAGGAGAG 2406 CUCUCCUAUGUGCUGGCCUUGGU 2426AD-20450.1 AAGGCCAGCACAUAGGAGAGA 2407 UCUCUCCUAUGUGCUGGCCUUGG 2427AD-20451.1 AGGCCAGCACAUAGGAGAGAU 2408 AUCUCUCCUAUGUGCUGGCCUUG 2428AD-20452.1 GGCCAGCACAUAGGAGAGAUG 2409 CAUCUCUCCUAUGUGCUGGCCUU 2429AD-20453.1 GCCAGCACAUAGGAGAGAUGA 2410 UCAUCUCUCCUAUGUGCUGGCCU 2430AD-20454.1 CCAGCACAUAGGAGAGAUGAG 2411 CUCAUCUCUCCUAUGUGCUGGCC 2431AD-20455.1 CAGCACAUAGGAGAGAUGAGC 2412 GCUCAUCUCUCCUAUGUGCUGGC 2432AD-20456.1 AGCACAUAGGAGAGAUGAGCU 2413 AGCUCAUCUCUCCUAUGUGCUGG 2433AD-20457.1 CACAUAGGAGAGAUGAGCUUC 2414 GAAGCUCAUCUCUCCUAUGUGCU 2434AD-20458.1 ACAUAGGAGAGAUGAGCUUCC 2415 GGAAGCUCAUCUCUCCUAUGUGC 2435AD-20459.1 CAUAGGAGAGAUGAGCUUCCU 2416 AGGAAGCUCAUCUCUCCUAUGUG 2436AD-20460.1 AUAGGAGAGAUGAGCUUCCUA 2417 UAGGAAGCUCAUCUCUCCUAUGU 2437AD-20461.1 UAGGAGAGAUGAGCUUCCUAC 2418 GUAGGAAGCUCAUCUCUCCUAUG 2438AD-20462.1 AGGAGAGAUGAGCUUCCUACA 2419 UGUAGGAAGCUCAUCUCUCCUAU 2439AD-20463.1 GGAGAGAUGAGCUUCCUACAG 2420 CUGUAGGAAGCUCAUCUCUCCUA 2440AD-20464.1 GAGAGAUGAGCUUCCUACAGC 2421 GCUGUAGGAAGCUCAUCUCUCCU 2441AD-20465.1 AGAGAUGAGCUUCCUACAGCA 2422 UGCUGUAGGAAGCUCAUCUCUCC 2442AD-20466.1 GAGAUGAGCUUCCUACAGCAC 2423 GUGCUGUAGGAAGCUCAUCUCUC 2443

Example 14 Inhibition of Eg5/KSP and VEGF Expression in Humans

A human subject is treated with a pharmaceutical composition, e.g.,ALNVSP02, having both a SNALP formulated dsRNA targeted to a Eg5/KSPgene and a SNALP formulated dsRNA targeted to a VEGF gene to inhibitexpression of the Eg5/KSP and VEGF genes.

A subject in need of treatment is selected or identified. The subjectcan be in need of cancer treatment, e.g., liver cancer.

At time zero, a suitable first dose of the composition is subcutaneouslyadministered to the subject. The composition is formulated as describedherein. After a period of time, the subject's condition is evaluated,e.g., by measurement of tumor growth, measuring serum AFP levels, andthe like. This measurement can be accompanied by a measurement ofEg5/KSP and/or VEGF expression in said subject, and/or the products ofthe successful siRNA-targeting of Eg5/KSP and/or VEGF mRNA. Otherrelevant criteria can also be measured. The number and strength of dosesare adjusted according to the subject's needs.

After treatment, the subject's condition is compared to the conditionexisting prior to the treatment, or relative to the condition of asimilarly afflicted but untreated subject.

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the present disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

1. A composition comprising a first double-stranded ribonucleic acid(dsRNA) for inhibiting the expression of a human kinesin family member11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expressionof a human VEGF in a cell, wherein: both said first and said seconddsRNA are formulated in a stable nucleic acid lipid particle (SNALP);said first dsRNA consists of a first sense strand and a first antisensestrand, and said first sense strand comprises a first sequence and saidfirst antisense strand comprises a second sequence complementary to atleast 15 contiguous nucleotides of SEQ ID NO:1311(5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence iscomplementary to said second sequence and wherein said first dsRNA isbetween 15 and 30 base pairs in length; and said second dsRNA consistsof a second sense strand and a second antisense strand, said secondsense strand comprising a third sequence and said second antisensestrand comprising a fourth sequence complementary to at least 15contiguous nucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′),wherein said third sequence is complementary to said fourth sequence andwherein each strand is between 15 and 30 base pairs in length.
 2. Thecomposition of claim 1, wherein the first antisense strand comprises asecond sequence complementary to SEQ ID NO:1311(5′-UCGAGAAUCUAAACUAACU-3′) and the second antisense strand comprises afourth sequence complementary to SEQ ID NO:1538(5′-GCACAUAGGAGAGAUGAGCUU-3′).
 3. The composition of claim 1, whereinthe first dsRNA consists of a sense strand consisting of SEQ ID NO:1534(5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQID NO:1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consistsof a sense strand consisting of SEQ ID NO:1536(5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQID NO:1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′).
 4. The composition of claim3, wherein each strand is modified as follows to include a 2′-O-methylribonucleotide as indicated by a lower case letter “c” or “u” and aphosphorothioate as indicated by a lower case letter “s”: the firstdsRNA consists of a sense strand consisting of SEQ ID NO: 1240(5′-ucGAGAAucuAAAcuAAcuTsT-3′)

and an antisense strand consisting of SEQ ID NO: 1241(5′-AGUuAGUUuAGAUUCUCGATsT);

the second dsRNA consists of a sense strand consisting of SEQ ID NO:1242 (5′-GcAcAuAGGAGAGAuGAGCUsU-3′)

and an antisense strand consisting of SEQ ID NO: 1243(5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).


5. The composition of claim 1, wherein said first and second dsRNAcomprises at least one modified nucleotide.
 6. The composition of claim5, wherein said modified nucleotide is chosen from the group of: a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 7. Thecomposition of claim 5, wherein said modified nucleotide is chosen fromthe group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide.
 8. The composition of claim 1, wherein said first and seconddsRNA each comprise at least one 2′-O-methyl modified ribonucleotide andat least one nucleotide comprising a 5′-phosphorothioate group.
 9. Thecomposition of claim 1, wherein each strand of each dsRNA is 19-23 basesin length.
 10. The composition of claim 1, wherein each strand of eachdsRNA is 21-23 bases in length.
 11. The composition of claim 1, whereineach strand of the first dsRNA is 21 bases in length and the sensestrand of the second dsRNA is 21 bases in length and the antisensestrand of the second dsRNA is 23 bases in length.
 12. The composition ofclaim 1, wherein the first and second dsRNA are present in an equimolarratio.
 13. The composition of claim 1, wherein said SNALP comprisesDLinDMA, cholesterol, DPPC, and PEG2000-C-DMA.
 14. The composition ofclaim 1, comprising the components in the proportions listed in Table17.
 15. The composition of claim 1, wherein said composition, uponcontact with a cell expressing Eg5, inhibits expression of Eg5 by atleast 40, 50, 60, 70, 80, or by at least 90%.
 16. The composition ofclaim 1, wherein said composition, upon contact with a cell expressingVEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or byat least 90%.
 17. The composition of claim 1, wherein administration ofsaid composition to a cell decreases expression of both Eg5 and VEGF insaid cell.
 18. The composition of claim 1, wherein the composition isadministered in a nM concentration.
 19. The composition of claim 1,wherein administration of said composition to a cell increasesmono-aster formation in the cell.
 20. The composition of claim 1,wherein administration of said composition to a mammal results in atleast one effect selected from the group consisting of prevention oftumor growth, reduction in tumor growth, or prolonged survival in saidmammal.
 21. The composition of claim 1, wherein said effect is measuredusing at least one assay selected from the group consisting ofdetermination of body weight, determination of organ weight, visualinspection, mRNA analysis, serum AFP analysis and survival monitoring.22. The composition of claim 1, further comprising Sorafenib.
 23. Thecomposition of claim 1, wherein the first dsRNA contains two overhangsand the second dsRNA contains an overhang at the 3′ of the antisense anda blunt end at the 5′ end of the antisense strand.
 24. A method forinhibiting the expression of Eg5/KSP and VEGF in a cell comprisingadministering any of the compositions of claim 1 to the cell.
 25. Amethod for preventing tumor growth, reducing tumor growth, or prolongingsurvival in a mammal in need of treatment for cancer comprisingadministering the composition of claim 1 to said mammal.
 26. The methodof claim 25, wherein said mammal has liver cancer.
 27. The method ofclaim 25, wherein said mammal is a human with liver cancer.
 28. Themethod of claim 24, further comprising administering Sorafenib.